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Sample records for naked poliovirus replicons

  1. Construction and characterization of poliovirus subgenomic replicons.

    PubMed

    Kaplan, G; Racaniello, V R

    1988-05-01

    Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2Apro, 2C, and 3Dpol and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. Replication of P2/Lansing virion RNA was inhibited by cotransfection with the R1 replicon, as demonstrated by hybridization analysis with a serotype-specific oligonucleotide probe. A higher level of inhibition of RNA replication was observed when P2/Lansing RNA was cotransfected into HeLa cells with truncated R1 transcripts (R1-PvuII) that were missing 395 3' nucleotides and a poly(A) tail. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs. No inhibition of plaque size was observed when replicon RNAs were used that were missing 1,384 or 1,839 3' nucleotides or contained plasmid

  2. Naked sunspots

    NASA Technical Reports Server (NTRS)

    Liggett, M.; Zirin, H.

    1983-01-01

    Naked sunspots are spots seen in H-alpha to be devoid of associated plage. In magnetograms and K-line little if any opposite polarity field is found, and in soft X ray images a blank appears in the region of the spot. In almost all cases studied in which naked spots resulted the spot groups had emerged in unipolar regions of the same polarity as the naked spot. At least half of the naked spots are associated with coronal holes. The naked spots are long-lived and show rotation rates close to the Newton-Nunn curve. Most of the naked spots had bright rims in H-alpha, and the one spot observed to disappear left no trace in the background magnetic field. These spots may be a means by which separation of p from f magnetic polarity occurs.

  3. 'Naked' radiopharmaceuticals

    SciTech Connect

    Wallner, Paul E. . E-mail: pwallner@rtsx.com

    2006-10-01

    The term 'naked' radiopharmaceuticals, more appropriately, 'unbound' radiopharmaceuticals, refers to any radioisotope used for clinical research or clinical purposes that is not attached to a chemical or biological carrier, and that localizes in various tissues because of a physiologic or chemical propensity/affinity, or secondary to focal anatomic placement. Although they remain useful in selected clinical circumstances, the available agents (except for Iodine-131) have been relegated to an unfortunate and somewhat secondary role. The agents remain useful and worthy of consideration for new clinical investigation and clinical use.

  4. Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors.

    PubMed

    Wang, Hai-Chao; An, Huai-Jie; Yu, Yun-Zhou; Xu, Qing

    2015-02-01

    DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax. PMID:25102364

  5. Vaccine-derived polioviruses.

    PubMed

    Burns, Cara C; Diop, Ousmane M; Sutter, Roland W; Kew, Olen M

    2014-11-01

    The attenuated oral poliovirus vaccine (OPV) has many properties favoring its use in polio eradication: ease of administration, efficient induction of intestinal immunity, induction of durable humoral immunity, and low cost. Despite these advantages, OPV has the disadvantage of genetic instability, resulting in rare and sporadic cases of vaccine-associated paralytic poliomyelitis (VAPP) and the emergence of genetically divergent vaccine-derived polioviruses (VDPVs). Whereas VAPP is an adverse event following exposure to OPV, VDPVs are polioviruses whose genetic properties indicate prolonged replication or transmission. Three categories of VDPVs are recognized: (1) circulating VDPVs (cVDPVs) from outbreaks in settings of low OPV coverage, (2) immunodeficiency-associated VDPVs (iVDPVs) from individuals with primary immunodeficiencies, and (3) ambiguous VDPVs (aVDPVs), which cannot be definitively assigned to either of the first 2 categories. Because most VDPVs are type 2, the World Health Organization's plans call for coordinated worldwide replacement of trivalent OPV with bivalent OPV containing poliovirus types 1 and 3. PMID:25316847

  6. Chromosomal replicons of higher plants

    SciTech Connect

    Van't Hof, J.

    1987-03-16

    This brief discussion of replicons of higher plants offers a glimpse into the properties of chromosomal DNA replication. It gives evidence that the S phase of unrelated plant species is comprised of temporally ordered replicon families that increase in number with genome size. This orderly process, which assures a normal inheritance of genetic material to recipient daughter cells, is maintained at the level of replicon clusters by two mutually exclusive mechanisms, one involving the rate at which single replicons replicate their allotment of DNA, and another by means of the tempo-pause. The same two mechanisms are used by cells to alter the pattern of chromosomal DNA replication just prior to and during normal development. Both mechanisms are genetically determined and produce genetic effects when disturbed of disrupted by additional non-conforming DNAs. Further insight into how these two mechanisms operate requires more molecular information about the nature of replicons and the factors that govern when a replicon family replicates. Plant material is a rich and ideal source for this information just awaiting exploitation. 63 refs.

  7. Phylogenetic Analysis of Poliovirus Sequences.

    PubMed

    Jorba, Jaume

    2016-01-01

    Comparative genomic sequencing is a major surveillance tool in the Polio Laboratory Network. Due to the rapid evolution of polioviruses (~1 % per year), pathways of virus transmission can be reconstructed from the pathways of genomic evolution. Here, we describe three main phylogenetic methods; estimation of genetic distances, reconstruction of a maximum-likelihood (ML) tree, and estimation of substitution rates using Bayesian Markov chain Monte Carlo (MCMC). The data set used consists of complete capsid sequences from a survey of poliovirus sequences available in GenBank. PMID:26983737

  8. Isolation and characterization of HeLa cell lines blocked at different steps in the poliovirus life cycle.

    PubMed Central

    Kaplan, G; Levy, A; Racaniello, V R

    1989-01-01

    Cotransfection of poliovirus RNA and R1, a poliovirus subgenomic RNA containing a deletion of nearly all of the capsid region, resulted in surviving cells, in contrast to the complete cell death observed after transfection with viral RNA. Cells that survived the cotransfection grew into colonies, produced infectious poliovirus, and underwent cycles of cell lysis (crisis periods) where less than 1% of the cells survived, followed by periods of growth. Poliovirus evolved during the persistent infection as judged by changes in plaque size. After passage for 6 months, a stable line called SOFIA emerged that no longer produced infectious virus and did not contain viral proteins or viral RNA. Cells frozen in liquid N2 while still in crisis and recultured 4 months later (named SOFIA N2) were also stabilized. After infection with poliovirus, SOFIA N2 cells showed a delay in the development of cytopathic effect, viral production, and cellular death when compared with HeLa cells. In contrast, SOFIA cells did not develop cytopathic effect and produced 10,000 times less virus than SOFIA N2 or HeLa cells. Viral production was delayed in SOFIA and SOFIA N2 cells transfected with poliovirus RNA when compared with HeLa cells, suggesting the presence of an intracellular block to poliovirus replication. Analysis of the cellular receptor for poliovirus by virus binding, an enzyme-linked immunosorbent assay, and in situ rosette assays with an antireceptor monoclonal antibody showed that receptors were expressed in SOFIA N2 cells but not in SOFIA cells. Echovirus 6, an enterovirus which uses a different cellular receptor, formed small plaques on SOFIA cells. Vesicular stomatitis virus formed plaques of similar size on SOFIA and HeLa cells, suggesting that the intracellular block was specific for enteroviruses. Cotransfection of the subgenomic replicon R1 with poliovirion RNA therefore resulted in the selection of HeLa cell variants containing blocks to poliovirus replication at the

  9. Timelike naked singularity

    SciTech Connect

    Goswami, Rituparno; Joshi, Pankaj S.; Vaz, Cenalo; Witten, Louis

    2004-10-15

    We construct a class of spherically symmetric collapse models in which a naked singularity may develop as the end state of collapse. The matter distribution considered has negative radial and tangential pressures, but the weak energy condition is obeyed throughout. The singularity forms at the center of the collapsing cloud and continues to be visible for a finite time. The duration of visibility depends on the nature of energy distribution. Hence the causal structure of the resulting singularity depends on the nature of the mass function chosen for the cloud. We present a general model in which the naked singularity formed is timelike, neither pointlike nor null. Our work represents a step toward clarifying the necessary conditions for the validity of the Cosmic Censorship Conjecture.

  10. Naked Black Hole Firewalls

    NASA Astrophysics Data System (ADS)

    Chen, Pisin; Ong, Yen Chin; Page, Don N.; Sasaki, Misao; Yeom, Dong-han

    2016-04-01

    In the firewall proposal, it is assumed that the firewall lies near the event horizon and should not be observable except by infalling observers, who are presumably terminated at the firewall. However, if the firewall is located near where the horizon would have been, based on the spacetime evolution up to that time, later quantum fluctuations of the Hawking emission rate can cause the "teleological" event horizon to have migrated to the inside of the firewall location, rendering the firewall naked. In principle, the firewall can be arbitrarily far outside the horizon. This casts doubt about the notion that firewalls are the "most conservative" solution to the information loss paradox.

  11. Naked Black Hole Firewalls.

    PubMed

    Chen, Pisin; Ong, Yen Chin; Page, Don N; Sasaki, Misao; Yeom, Dong-Han

    2016-04-22

    In the firewall proposal, it is assumed that the firewall lies near the event horizon and should not be observable except by infalling observers, who are presumably terminated at the firewall. However, if the firewall is located near where the horizon would have been, based on the spacetime evolution up to that time, later quantum fluctuations of the Hawking emission rate can cause the "teleological" event horizon to have migrated to the inside of the firewall location, rendering the firewall naked. In principle, the firewall can be arbitrarily far outside the horizon. This casts doubt about the notion that firewalls are the "most conservative" solution to the information loss paradox. PMID:27152788

  12. Characterization of cell lines stably transfected with rubella virus replicons

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  13. Current status of poliovirus infections.

    PubMed Central

    Melnick, J L

    1996-01-01

    Two scientists who played leading roles in the conquest of poliomyelitis died recently. In 1954, Jonas Salk provided the first licensed polio vaccine, the formalin (and heat)-inactivated virus. Albert Sabin gave us the attenuated live virus vaccine, which was licensed in 1962. This paper takes the reader through the history of the disease, including its pathogenesis, epidemiology, vaccines, and future directions. The emphasis is on vaccines, for it seems that with proper vaccination the number of new cases is falling dramatically. It is hoped that by the year 2000, we will accomplish the goal of the World Health Organization of "a world without polio." Then, because there is no animal reservoir, we can seriously discuss when and how to eliminate the need for vaccination and ultimately destroy our stocks of poliovirus. PMID:8809461

  14. Current status of poliovirus infections.

    PubMed

    Melnick, J L

    1996-07-01

    Two scientists who played leading roles in the conquest of poliomyelitis died recently. In 1954, Jonas Salk provided the first licensed polio vaccine, the formalin (and heat)-inactivated virus. Albert Sabin gave us the attenuated live virus vaccine, which was licensed in 1962. This paper takes the reader through the history of the disease, including its pathogenesis, epidemiology, vaccines, and future directions. The emphasis is on vaccines, for it seems that with proper vaccination the number of new cases is falling dramatically. It is hoped that by the year 2000, we will accomplish the goal of the World Health Organization of "a world without polio." Then, because there is no animal reservoir, we can seriously discuss when and how to eliminate the need for vaccination and ultimately destroy our stocks of poliovirus. PMID:8809461

  15. Inhibition of poliovirus RNA synthesis by brefeldin A.

    PubMed Central

    Maynell, L A; Kirkegaard, K; Klymkowsky, M W

    1992-01-01

    Brefeldin A (BFA), a fungal metabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was found to inhibit poliovirus replication 10(5)- to 10(6)-fold. BFA does not inhibit entry of poliovirus into the cell or translation of viral RNA. Poliovirus RNA synthesis, however, is completely inhibited by BFA. A specific class of membranous vesicles, with which the poliovirus replication complex is physically associated, is known to proliferate in poliovirus-infected cells. BFA may inhibit poliovirus replication by preventing the formation of these vesicles. Images PMID:1312615

  16. Will containment of wild poliovirus in laboratories and inactivated poliovirus vaccine production sites be effective for global certification?

    PubMed Central

    Dowdle, Walter R.; Wolff, Christopher; Sanders, Raymond; Lambert, Scott; Best, Maureen

    2004-01-01

    The absolute laboratory containment of any virus cannot be guaranteed, but a wealth of experience indicates that effective containment of wild poliovirus materials for global certification is technically and operationally feasible. Effective containment is based on the principles of minimal wild poliovirus infectious and potentially infectious materials in laboratories; minimal risks of operations in laboratories and inactivated poliovirus vaccine production facilities; minimal susceptibility of workers to wild poliovirus infection and shedding; and minimal susceptibility of populations to wild poliovirus spread. Each principle alone is imperfect, but collectively they greatly minimize the risks of transmitting wild poliovirus from the laboratory to the community. PMID:15106302

  17. Model-driven engineering of gene expression from RNA replicons.

    PubMed

    Beal, Jacob; Wagner, Tyler E; Kitada, Tasuku; Azizgolshani, Odisse; Parker, Jordan Moberg; Densmore, Douglas; Weiss, Ron

    2015-01-16

    RNA replicons are an emerging platform for engineering synthetic biological systems. Replicons self-amplify, can provide persistent high-level expression of proteins even from a small initial dose, and, unlike DNA vectors, pose minimal risk of chromosomal integration. However, no quantitative model sufficient for engineering levels of protein expression from such replicon systems currently exists. Here, we aim to enable the engineering of multigene expression from more than one species of replicon by creating a computational model based on our experimental observations of the expression dynamics in single- and multireplicon systems. To this end, we studied fluorescent protein expression in baby hamster kidney (BHK-21) cells using a replicon derived from Sindbis virus (SINV). We characterized expression dynamics for this platform based on the dose-response of a single species of replicon over 50 h and on a titration of two cotransfected replicons expressing different fluorescent proteins. From this data, we derive a quantitative model of multireplicon expression and validate it by designing a variety of three-replicon systems, with profiles that match desired expression levels. We achieved a mean error of 1.7-fold on a 1000-fold range, thus demonstrating how our model can be applied to precisely control expression levels of each Sindbis replicon species in a system. PMID:24877739

  18. Poliovirus Adsorption by 34 Minerals and Soils

    PubMed Central

    Moore, Rebecca S.; Taylor, Dene H.; Sturman, Lawrence S.; Reddy, Michael M.; Fuhs, G. Wolfgang

    1981-01-01

    The adsorption of radiolabeled infectious poliovirus type 2 by 34 well-defined soils and mineral substrates was analyzed in a synthetic freshwater medium containing 1 mM CaCl2 and 1.25 mM NaHCO3 at pH 7. In a model system, adsorption of poliovirus by Ottawa sand was rapid and reached equilibrium within 1 h at 4°C. Near saturation, the adsorption could be described by the Langmuir equation; the apparent surface saturation was 2.5 × 106 plaque-forming units of poliovirus per mg of Ottawa sand. At low surface coverage, adsorption was described by the Freundlich equation. The soils and minerals used ranged from acidic to basic and from high in organic content to organic free. The available negative surface charge on each substrate was measured by the adsorption of a cationic polyelectrolyte, polydiallyldimethylammonium chloride. Most of the substrates adsorbed more than 95% of the virus. In general, soils, in comparison with minerals, were weak adsorbents. Among the soils, muck and Genesee silt loam were the poorest adsorbents; among the minerals, montmorillonite, glauconite, and bituminous shale were the least effective. The most effective adsorbents were magnetite sand and hematite, which are predominantly oxides of iron. Correlation coefficients for substrate properties and virus adsorption revealed that the elemental composition of the adsorbents had little effect on poliovirus uptake. Substrate surface area and pH, by themselves, were not significantly correlated with poliovirus uptake. A strong negative correlation was found between poliovirus adsorption and both the contents of organic matter and the available negative surface charge on the substrates as determined by their capacities for adsorbing the cationic polyelectrolyte, polydiallyldimethylammonium chloride. PMID:6274259

  19. Poliovirus: Generation and Characterization of Mutants

    PubMed Central

    Burrill, Cecily P.; Strings, Vanessa R.; Schulte, Michael B.; Andino, Raul

    2016-01-01

    Poliovirus (PV) is the prototypical picornavirus. It is a non-enveloped RNA virus with a small (~7.5 kb) genome of positive polarity. cDNA clones of several strains are available, and infectious virus can be produced by the transfection of in vitro transcribed viral genomes into an appropriate host cell. The ease of genetic studies in poliovirus is a primary reason that it has long served as a model to study RNA virus biology, pathogenesis, and evolution. Protocols for the generation and characterization of PV mutants are presented. A separate unit concerning the production, propagation, quantification, and purification of PV will also be presented. PMID:23686829

  20. Visualization of DNA replicons by image cytometry

    SciTech Connect

    Gratzner, H.G. )

    1993-01-01

    Replication of DNA in eukaryotic organisms proceeds bidirectionally along the double helix in replicon substructures. The process can be visualized by autoradiography of spreads of genomic DNA from lysed, whole cells which have been incubated with radioactive DNA precursors. The objective of the present study was to develop techniques to measure DNA strand initiation and elongation using immunofluorescence and image cytometry. Peripheral blood lymphocytes were cultured for 3 days with PHA and then pulsed for 5 or 15 minutes with iododeoxyuridine. Chromatin spreads were then produced on microscope slides by lysing with detergent and slides were immunofluorescently stained by an indirect anti-BrdU technique. Individual replicons of mammalian cells were visualized by immunofluorescence at high resolution and digital images were analyzed to determine the rates of elongation as well as initiation parameters. Elongation rates by the method were approximately 1 [mu]M/min. The methods are applicable to visualization and quantitation of the effects of radiation or other agents on DNA damage or repair.

  1. Naked singularities as particle accelerators

    SciTech Connect

    Patil, Mandar; Joshi, Pankaj S.

    2010-11-15

    We investigate here the particle acceleration by naked singularities to arbitrarily high center of mass energies. Recently it has been suggested that black holes could be used as particle accelerators to probe the Planck scale physics. We show that the naked singularities serve the same purpose and probably would do better than their black hole counterparts. We focus on the scenario of a self-similar gravitational collapse starting from a regular initial data, leading to the formation of a globally naked singularity. It is seen that when particles moving along timelike geodesics interact and collide near the Cauchy horizon, the energy of collision in the center of mass frame will be arbitrarily high, thus offering a window to Planck scale physics.

  2. Poliovirus Laboratory Based Surveillance: An Overview.

    PubMed

    Zaidi, Syed Sohail Zahoor; Asghar, Humayun; Sharif, Salmaan; Alam, Muhammad Masroor

    2016-01-01

    World Health Assembly (WHA) in 1988 encouraged the member states to launch Global Polio Eradication Initiative (GPEI) (resolution WHA41.28) against "the Crippler" called poliovirus, through strong routine immunization program and intensified surveillance systems. Since its launch, global incidence of poliomyelitis has been reduced by more than 99 % and the disease squeezed to only three endemic countries (Afghanistan, Pakistan, and Nigeria) out of 125. Today, poliomyelitis is on the verge of eradication, and their etiological agents, the three poliovirus serotypes, are on the brink of extinction from the natural environment. The last case of poliomyelitis due to wild type 2 strain occurred in 1999 in Uttar Pradesh, India whereas the last paralytic case due to wild poliovirus type 3 (WPV3) was seen in November, 2012 in Yobe, Nigeria. Despite this progress, undetected circulation cannot fully rule out the eradication as most of the poliovirus infections are entirely subclinical; hence sophisticated environmental surveillance is needed to ensure the complete eradication of virus. Moreover, the vaccine virus in under-immunized communities can sometimes revert and attain wild type characteristics posing a big challenge to the program. PMID:26983728

  3. MAGNETIC TOPOLOGY OF A NAKED SUNSPOT: IS IT REALLY NAKED?

    SciTech Connect

    Sainz Dalda, A.; Vargas Dominguez, S.; Tarbell, T. D.

    2012-02-10

    The high spatial, temporal, and spectral resolution achieved by Hinode instruments gives much better understanding of the behavior of some elusive solar features, such as pores and naked sunspots. Their fast evolution and, in some cases, their small sizes have made their study difficult. The moving magnetic features (MMFs) have been studied during the last 40 years. They have been always associated with sunspots, especially with the penumbra. However, a recent observation of a naked sunspot (one with no penumbra) has shown MMF activity. The authors of this reported observation expressed their reservations about the explanation given to the bipolar MMF activity as an extension of the penumbral filaments into the moat. How can this type of MMF exist when a penumbra does not? In this Letter, we study the full magnetic and (horizontal) velocity topology of the same naked sunspot, showing how the existence of a magnetic field topology similar to that observed in sunspots can explain these MMFs, even when the intensity map of the naked sunspot does not show a penumbra.

  4. Transgenic mice carrying the human poliovirus receptor: new animal models for study of poliovirus neurovirulence.

    PubMed Central

    Horie, H; Koike, S; Kurata, T; Sato-Yoshida, Y; Ise, I; Ota, Y; Abe, S; Hioki, K; Kato, H; Taya, C

    1994-01-01

    Recombinant viruses between the virulent Mahoney and attenuated Sabin 1 strains of poliovirus type 1 were subjected to neurovirulence tests using a transgenic (Tg) mouse line, ICR-PVRTg1, that carried the human poliovirus receptor gene. The Tg mice were inoculated intracerebrally with these recombinant viruses and observed for clinical signs, histopathological lesions, and viral antigens as parameters of neurovirulence of the viruses. These parameters observed in the Tg mice were different for different inoculated viruses. Dose-dependent incidences of paralysis and of death were observed in the Tg mice inoculated with any viruses used. This indicates that values of 50% lethal dose are useful to score a wide range of neurovirulence of poliovirus. The neurovirulence of individual viruses estimated by the Tg mouse model had a strong correlation with those estimated by monkey model. Consequently, the mouse tests identified the neurovirulence determinants on the genome of poliovirus that had been identified by monkey tests. In addition, the mouse tests revealed new neurovirulence determinants, that is, different nucleotides between the two strains at positions 189 and 21 and/or 935 in the 5'-proximal 1,122 nucleotides. The Tg mice used in this study may be suitable for replacing monkeys for investigating poliovirus neurovirulence. Images PMID:8289371

  5. Shadow of a naked singularity

    NASA Astrophysics Data System (ADS)

    Ortiz, Néstor; Sarbach, Olivier; Zannias, Thomas

    2015-08-01

    We analyze the redshift suffered by photons originating from an external source, traversing a collapsing dust cloud, and finally being received by an asymptotic observer. In addition, we study the shadow that the collapsing cloud casts on the sky of the asymptotic observer. We find that the resulting redshift and properties of the shadow depend crucially on whether the final outcome of the complete gravitational collapse is a black hole or a naked singularity. In the black hole case, the shadow is due to the high redshift acquired by the photons as they approach the event horizon, implying that their energy is gradually redshifted toward zero within a few crossing times associated with the event horizon radius. In contrast to this, a naked singularity not only absorbs photons originating from the source, but it also emits infinitely redshifted photons with and without angular momenta. This emission introduces an abrupt cutoff in the frequency shift of the photons detected in directions close to the radial one, and it is responsible for the shadow masking the source in the naked singularity case. Furthermore, even though the shadow forms and begins to grow immediately after the observer crosses the Cauchy horizon, it takes many more crossing times than in the black hole case for the source to be occulted from the observer's eyes. We discuss possible implications of our results for testing the weak cosmic censorship hypothesis. Even though at late times the image of the source perceived by the observer looks the same in both cases, the dynamical formation of the shadow and the redshift images has distinct features and time scales in the black hole versus the naked singularity case. For stellar collapse, these time scales seem to be too short to be resolved with existing technology. However, our results may be relevant for the collapse of seeds leading to supermassive black holes.

  6. Cessation of Trivalent Oral Poliovirus Vaccine and Introduction of Inactivated Poliovirus Vaccine - Worldwide, 2016.

    PubMed

    Hampton, Lee M; Farrell, Margaret; Ramirez-Gonzalez, Alejandro; Menning, Lisa; Shendale, Stephanie; Lewis, Ian; Rubin, Jennifer; Garon, Julie; Harris, Jennifer; Hyde, Terri; Wassilak, Steven; Patel, Manish; Nandy, Robin; Chang-Blanc, Diana

    2016-01-01

    Since the 1988 World Health Assembly resolution to eradicate poliomyelitis, transmission of the three types of wild poliovirus (WPV) has been sharply reduced (1). WPV type 2 (WPV2) has not been detected since 1999 and was declared eradicated in September 2015. Because WPV type 3 has not been detected since November 2012, WPV type 1 (WPV1) is likely the only WPV that remains in circulation (1). This marked progress has been achieved through widespread use of oral poliovirus vaccines (OPVs), most commonly trivalent OPV (tOPV), which contains types 1, 2, and 3 live, attenuated polioviruses and has been a mainstay of efforts to prevent polio since the early 1960s. However, attenuated polioviruses in OPV can undergo genetic changes during replication, and in communities with low vaccination coverage, can result in vaccine-derived polioviruses (VDPVs) that can cause paralytic polio indistinguishable from the disease caused by WPVs (2). Among the 721 polio cases caused by circulating VDPVs (cVDPVs*) detected during January 2006-May 2016, type 2 cVDPVs (cVDPV2s) accounted for >94% (2). Eliminating the risk for polio caused by VDPVs will require stopping all OPV use. The first stage of OPV withdrawal involved a global, synchronized replacement of tOPV with bivalent OPV (bOPV) containing only types 1 and 3 attenuated polioviruses, planned for April 18-May 1, 2016, thereby withdrawing OPV type 2 from all immunization activities (3). Complementing the switch from tOPV to bOPV, introduction of at least 1 dose of injectable, trivalent inactivated poliovirus vaccine (IPV) into childhood immunization schedules reduces risks from and facilitates responses to cVDPV2 outbreaks. All 155 countries and territories that were still using OPV in immunization schedules in 2015 have reported that they had ceased use of tOPV by mid-May 2016.(†) As of August 31, 2016, 173 (89%) of 194 World Health Organization (WHO) countries included IPV in their immunization schedules.(§) The cessation of

  7. Multiplex PCR Method for Identifying Recombinant Vaccine-Related Polioviruses

    PubMed Central

    Kilpatrick, David R.; Ching, Karen; Iber, Jane; Campagnoli, Ray; Freeman, Christopher J.; Mishrik, Nada; Liu, Hong-Mei; Pallansch, Mark A.; Kew, Olen M.

    2004-01-01

    The recent discovery of recombinant circulating vaccine-derived poliovirus (recombinant cVDPV) has highlighted the need for enhanced global poliovirus surveillance to assure timely detection of any future cVDPV outbreaks. Six pairs of Sabin strain-specific recombinant primers were designed to permit rapid screening for VDPV recombinants by PCR. PMID:15365031

  8. Multiple replicons constituting the genome of Pseudomonas cepacia 17616.

    PubMed Central

    Cheng, H P; Lessie, T G

    1994-01-01

    Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 17616, in conjunction with Southern hybridization experiments using junction fragments containing rare restriction enzyme sites as probes, indicated that this bacterium contains three large circular replicons of 3.4, 2.5, and 0.9 megabases (Mb). Inclusion of the 170-kb cryptic plasmid present in this strain gave an overall estimate of genome size of 7 Mb. Other Southern hybridization experiments indicated that the three large replicons contained rRNA genes as well as insertion sequence elements identified previously in this strain. The distribution of SwaI, PacI, and PmeI sites on the three replicons was determined. A derivative of Tn5-751 carrying a SwaI site was used to inactivate and map genes on the 2.5- and 3.4-Mb replicons. Mutants were isolated in which the 2.5- and 0.9-Mb replicons had been reduced in size to 1.8 and 0.65 Mb, respectively. The loss of DNA from the 2.5-Mb replicon was associated with lysine auxotrophy, beta-lactamase deficiency, and failure to utilize ribitol and trehalose as carbon and energy sources. DNA fragments corresponding in size to randomly linearized forms of the different replicons were detected in unrestricted DNA by pulsed-field gel electrophoresis. The results provide a framework for further genetic analysis of strain 17616 and for evaluation of the genomic complexities of other P. cepacia isolates. Images PMID:7517389

  9. Plasmid replicon typing of commensal and pathogenic Escherichia coli isolates.

    PubMed

    Johnson, Timothy J; Wannemuehler, Yvonne M; Johnson, Sara J; Logue, Catherine M; White, David G; Doetkott, Curt; Nolan, Lisa K

    2007-03-01

    Despite the critical role of plasmids in horizontal gene transfer, few studies have characterized plasmid relatedness among different bacterial populations. Recently, a multiplex PCR replicon typing protocol was developed for classification of plasmids occurring in members of the Enterobacteriaceae. Here, a simplified version of this replicon typing procedure which requires only three multiplex panels to identify 18 plasmid replicons is described. This method was used to screen 1,015 Escherichia coli isolates of avian, human, and poultry meat origin for plasmid replicon types. Additionally, the isolates were assessed for their content of several colicin-associated genes. Overall, a high degree of plasmid variability was observed, with 221 different profiles occurring among the 1,015 isolates examined. IncFIB plasmids were the most common type identified, regardless of the source type of E. coli. IncFIB plasmids occurred significantly more often in avian pathogenic E. coli (APEC) and retail poultry E. coli (RPEC) than in uropathogenic E. coli (UPEC) and avian and human fecal commensal E. coli isolates (AFEC and HFEC, respectively). APEC and RPEC were also significantly more likely than UPEC, HFEC, and AFEC to possess the colicin-associated genes cvaC, cbi, and/or cma in conjunction with one or more plasmid replicons. The results suggest that E. coli isolates contaminating retail poultry are notably similar to APEC with regard to plasmid profiles, with both generally containing multiple plasmid replicon types in conjunction with colicin-related genes. In contrast, UPEC and human and avian commensal E. coli isolates generally lack the plasmid replicons and colicin-related genes seen in APEC and RPEC, suggesting limited dissemination of such plasmids among these bacterial populations. PMID:17277222

  10. Environmental Isolation of Circulating Vaccine-Derived Poliovirus After Interruption of Wild Poliovirus Transmission - Nigeria, 2016.

    PubMed

    Etsano, Andrew; Damisa, Eunice; Shuaib, Faisal; Nganda, Gatei Wa; Enemaku, Ogu; Usman, Samuel; Adeniji, Adekunle; Jorba, Jaume; Iber, Jane; Ohuabunwo, Chima; Nnadi, Chimeremma; Wiesen, Eric

    2016-01-01

    In September 2015, more than 1 year after reporting its last wild poliovirus (WPV) case in July 2014 (1), Nigeria was removed from the list of countries with endemic poliovirus transmission,* leaving Afghanistan and Pakistan as the only remaining countries with endemic WPV. However, on April 29, 2016, a laboratory-confirmed, circulating vaccine-derived poliovirus type 2 (cVDPV2) isolate was reported from an environmental sample collected in March from a sewage effluent site in Maiduguri Municipal Council, Borno State, a security-compromised area in northeastern Nigeria. VDPVs are genetic variants of the vaccine viruses with the potential to cause paralysis and can circulate in areas with low population immunity. The Nigeria National Polio Emergency Operations Center initiated emergency response activities, including administration of at least 2 doses of oral poliovirus vaccine (OPV) to all children aged <5 years through mass campaigns; retroactive searches for missed cases of acute flaccid paralysis (AFP), and enhanced environmental surveillance. Approximately 1 million children were vaccinated in the first OPV round. Thirteen previously unreported AFP cases were identified. Enhanced environmental surveillance has not resulted in detection of additional VDPV isolates. The detection of persistent circulation of VDPV2 in Borno State highlights the low population immunity, surveillance limitations, and risk for international spread of cVDPVs associated with insurgency-related insecurity. Increasing vaccination coverage with additional targeted supplemental immunization activities and reestablishment of effective routine immunization activities in newly secured and difficult-to-reach areas in Borno is urgently needed. PMID:27490081

  11. Naked singularity resolution in cylindrical collapse

    SciTech Connect

    Kurita, Yasunari; Nakao, Ken-ichi

    2006-03-15

    In this paper, we study the gravitational collapse of null dust in cylindrically symmetric spacetime. The naked singularity necessarily forms at the symmetry axis. We consider the situation in which null dust is emitted again from the naked singularity formed by the collapsed null dust and investigate the backreaction by this emission for the naked singularity. We show a very peculiar but physically important case in which the same amount of null dust as that of the collapsed one is emitted from the naked singularity as soon as the ingoing null dust hits the symmetry axis and forms the naked singularity. In this case, although this naked singularity satisfies the strong curvature condition by Krolak (limiting focusing condition), geodesics which hit the singularity can be extended uniquely across the singularity. Therefore, we may say that the collapsing null dust passes through the singularity formed by itself and then leaves for infinity. Finally, the singularity completely disappears and the flat spacetime remains.

  12. Quantum dress for a naked singularity

    NASA Astrophysics Data System (ADS)

    Casals, Marc; Fabbri, Alessandro; Martínez, Cristián; Zanelli, Jorge

    2016-09-01

    We investigate semiclassical backreaction on a conical naked singularity space-time with a negative cosmological constant in (2 + 1)-dimensions. In particular, we calculate the renormalized quantum stress-energy tensor for a conformally coupled scalar field on such naked singularity space-time. We then obtain the backreacted metric via the semiclassical Einstein equations. We show that, in the regime where the semiclassical approximation can be trusted, backreaction dresses the naked singularity with an event horizon, thus enforcing (weak) cosmic censorship.

  13. Naked singularities in initial surfaces

    SciTech Connect

    Wojtkiewicz, J. )

    1990-03-15

    We consider a singular hypersurface {Sigma}, carrying time-symmetric initial data for the Einstein equations. We assume that the area of the arbitrary two-sphere, enclosing the singularity, is bounded from below by some positive constant. A conformally flat ring,'' or pancake'' singularities having sufficiently large Euclidean radius, can serve as examples. We prove that if the Arnowitt-Deser-Misner mass associated with such a hypersurface is small enough, then this singularity is naked (i.e., it is not entirely surrounded by an apparent horizon). We suggest that a similar effect appears also for general (i.e., non-time-symmetric) hypersurfaces.

  14. Naked singularities and quantum gravity

    SciTech Connect

    Harada, Tomohiro; Iguchi, Hideo; Nakao, Ken-ichi; Singh, T. P.; Tanaka, Takahiro; Vaz, Cenalo

    2001-08-15

    There are known models of spherical gravitational collapse in which the collapse ends in a naked shell-focusing singularity for some initial data. If a massless scalar field is quantized on the classical background provided by such a star, it is found that the outgoing quantum flux of the scalar field diverges in the approach to the Cauchy horizon. We argue that the semiclassical approximation (i.e., quantum field theory on a classical curved background) used in these analyses ceases to be valid about one Planck time before the epoch of naked singularity formation, because by then the curvature in the central region of the star reaches the Planck scale. It is shown that during the epoch in which the semiclassical approximation is valid, the total emitted energy is about one Planck unit, and is not divergent. We also argue that back reaction in this model does not become important so long as gravity can be treated classically. It follows that the further evolution of the star will be determined by quantum gravitational effects, and without invoking quantum gravity it is not possible to say whether the star radiates away on a short time scale or settles down into a black hole state.

  15. Circulating vaccine-derived polioviruses: current state of knowledge.

    PubMed Central

    Kew, Olen M.; Wright, Peter F.; Agol, Vadim I.; Delpeyroux, Francis; Shimizu, Hiroyuki; Nathanson, Neal; Pallansch, Mark A.

    2004-01-01

    Within the past 4 years, poliomyelitis outbreaks associated with circulating vaccine-derived polioviruses (cVDPVs) have occurred in Hispaniola (2000-01), the Philippines (2001), and Madagascar (2001-02). Retrospective studies have also detected the circulation of endemic cVDPV in Egypt (1988-93) and the likely localized spread of oral poliovirus vaccine (OPV)-derived virus in Belarus (1965-66). Gaps in OPV coverage and the previous eradication of the corresponding serotype of indigenous wild poliovirus were the critical risk factors for all cVDPV outbreaks. The cVDPV outbreaks were stopped by mass immunization campaigns using OPV. To increase sensitivity for detecting vaccine-derived polioviruses (VDPVs), in 2001 the Global Polio Laboratory Network implemented additional testing requirements for all poliovirus isolates under investigation. This approach quickly led to the recognition of the Philippines and Madagascar cVDPV outbreaks, but of no other current outbreaks. The potential risk of cVDPV emergence has increased dramatically in recent years as wild poliovirus circulation has ceased in most of the world. The risk appears highest for the type 2 OPV strain because of its greater tendency to spread to contacts. The emergence of cVDPVs underscores the critical importance of eliminating the last pockets of wild poliovirus circulation, maintaining universally high levels of polio vaccine coverage, stopping OPV use as soon as it is safely possible to do so, and continuing sensitive poliovirus surveillance into the foreseeable future. Particular attention must be given to areas where the risks for wild poliovirus circulation have been highest, and where the highest rates of polio vaccine coverage must be maintained to suppress cVDPV emergence. PMID:15106296

  16. Seroimmunity to polioviruses in U.S. Army recruits.

    PubMed

    Burke, D S; Gaydos, J C; Hodder, R A; Bancroft, W H

    1979-02-01

    Titers of neutralizing antibody to poliovirus types 1, 2, and 3 were determined for serum specimens obtained from 268 U.S. Army recruits. Among those tested, 20.9% lacked neutralizing antibody to one or more types of poliovirus, and 1.1% lacked antibody to all three types. An analysis of demographic data showed that age of less than 18 years, schooling for less than 10 years, and residence in the northeastern United States were associated with higher percentages of recruits lacking neutralizing antibodies to polioviruses in serum. PMID:220335

  17. Concentration and purification of poliovirus by ultrafiltration and isopycnic centrifugation.

    PubMed

    Guskey, L E; Wolff, D A

    1972-07-01

    A method is described by which poliovirus can be rapidly and simply concentrated by the use of a Diaflo XM-50 ultrafilter membrane. Freon-extracted ultrafilter concentrates banded in CsCl provided a 1,724-fold volumetric concentration of poliovirus. During concentration, trypsin-digested cellular material can pass through the ultrafilter membrane, thus providing a versatile means of degrading and eliminating extraneous contaminating proteins. The ultrafilter concentration system is compared with the CsCl cushion system of poliovirus concentration, and both systems are further compared by banding virus and virus capsid material in CsCl by use of isopycnic centrifugation. PMID:4341520

  18. Naked singularity and a thunderbolt

    NASA Astrophysics Data System (ADS)

    Ishibashi, Akihiro; Hosoya, Akio

    2002-11-01

    We consider the quantum theoretical effects of a sudden change of boundary conditions which mimics the occurrence of naked singularities. For a simple demonstration, we study a massless scalar field in (1+1)-dimensional Minkowski spacetime with a finite spatial interval. We calculate the vacuum expectation value of the energy-momentum tensor and explicitly show that singular wave or thunderbolt appears along the Cauchy horizon. The thunderbolt possibly destroys the Cauchy horizon if its back reaction on the geometry is taken into account, leading to quantum restoration of the global hyperbolicity. The result of the present work may also apply to the situation where a closed string freely oscillating is traveling to a brane and changes itself to an open string pinned down by the ends, satisfying the Dirichlet boundary conditions on the brane.

  19. MULTIPLE REPLICONS CONSTITUTING THE GENOME OF PSEUDOMONAS CEPACIA 17616

    EPA Science Inventory

    Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 17616, in conjunction with Southern hybridization experiments using junction n enzyme sites as probes, indicated that this bacterium contains three large circular replicons of 3.4, 2.5, and 0.9 170-kb cryptic plas...

  20. Exploring the fitness landscape of poliovirus

    NASA Astrophysics Data System (ADS)

    Bianco, Simone; Acevedo, Ashely; Andino, Raul; Tang, Chao

    2012-02-01

    RNA viruses are known to display extraordinary adaptation capabilities to different environments, due to high mutation rates. Their very dynamical evolution is captured by the quasispecies concept, according to which the viral population forms a swarm of genetic variants linked through mutation, which cooperatively interact at a functional level and collectively contribute to the characteristics of the population. The description of the viral fitness landscape becomes paramount towards a more thorough understanding of the virus evolution and spread. The high mutation rate, together with the cooperative nature of the quasispecies, makes it particularly challenging to explore its fitness landscape. I will present an investigation of the dynamical properties of poliovirus fitness landscape, through both the adoption of new experimental techniques and theoretical models.

  1. Structural organization of replicon domains during DNA synthetic phase in the mammalian nucleus

    SciTech Connect

    Nakamura, H.; Morita, T.; Sato, C.

    1986-01-01

    In mammalian cells, it has been shown that adjacent multiple DNA replicons, termed a replicon cluster or a replicon domain, are replicated coordinately in a defined temporal order during the DNA synthetic (S) phase. However, no intranuclear structure of this replicon domain has been revealed in the nucleus labelled with (/sup 3/H)thymidine at the limited resolution level of autoradiography. Bu immunofluorescent staining with antibody against 5-bromodeoxyuridine (BrdU), we succeeded in detecting novel, intranuclear ring-like structures of replicating replicon domains that were organized temporarily during the S phase of mammalian cells with incorporated BrdU.

  2. Importation and circulation of poliovirus in Bulgaria in 2001.

    PubMed Central

    Kojouharova, Mira; Zuber, Patrick L. F.; Gyurova, Snejana; Fiore, Lucia; Buttinelli, Gabriele; Kunchev, Angel; Vladimirova, Nadejda; Korsun, Neli; Filipova, Radosveta; Boneva, Roumiana; Gavrilin, Eugene; Deshpande, Jagadish M.; Oblapenko, George; Wassilak, Steven G.

    2003-01-01

    OBJECTIVE: To characterize the circumstances in which poliomyelitis occurred among three children in Bulgaria during 2001 and to describe the public health response. METHODS: Bulgarian authorities investigated the three cases of polio and their contacts, conducted faecal and serological screening of children from high-risk groups, implemented enhanced surveillance for acute flaccid paralysis, and conducted supplemental immunization activities. FINDINGS: The three cases of polio studied had not been vaccinated and lived in socioeconomically deprived areas of two cities. Four Roma children from the Bourgas district had antibody titres to serotype 1 poliovirus only, and wild type 1 virus was isolated from the faeces of two asymptomatic Roma children in the Bourgas and Sofia districts. Poliovirus isolates were related genetically and represented a single evolutionary lineage; genomic sequences were less than 90% identical to poliovirus strains isolated previously in Europe, but 98.3% similar to a strain isolated in India in 2000. No cases or wild virus isolates were found after supplemental immunization activities were launched in May 2001. CONCLUSIONS: In Bulgaria, an imported poliovirus was able to circulate for two to five months among minority populations. Surveillance data strongly suggest that wild poliovirus circulation ceased shortly after supplemental immunization activities with oral poliovirus vaccine were conducted. PMID:12973639

  3. Morphine Enhances Hepatitis C Virus (HCV) Replicon Expression

    PubMed Central

    Li, Yuan; Zhang, Ting; Douglas, Steven D.; Lai, Jian-Ping; Xiao, Wei-Dong; Pleasure, David E.; Ho, Wen-Zhe

    2003-01-01

    Little information is available regarding whether substance abuse enhances hepatitis C virus (HCV) replication and promotes HCV disease progression. We investigated whether morphine alters HCV mRNA expression in HCV replicon-containing liver cells. Morphine significantly increased HCV mRNA expression, an effect which could be abolished by either of the opioid receptor antagonists, naltrexone or β-funaltrexamine. Investigation of the mechanism responsible for this enhancement of HCV replicon expression demonstrated that morphine activated NF-κB promoter and that caffeic acid phenethyl ester, a specific inhibitor of the activation of NF-κB, blocked morphine-activated HCV RNA expression. In addition, morphine compromised the anti-HCV effect of interferon alpha (IFN-α). Our in vitro data indicate that morphine may play an important role as a positive regulator of HCV replication in human hepatic cells and may compromise IFN-α therapy. PMID:12937158

  4. The replicon of pSW800 from Pantoea stewartii.

    PubMed

    Wu, C Y; Fu, J F; Liu, S T

    2001-10-01

    A 2019 bp DNA fragment containing the replicon of pSW800 from Pantoea stewartii SW2 was cloned and characterized. This replicon contains two genes--repA and repB, which encode a 36.5 kDa replication initiation protein (RepA) and a peptide of 18 aa, respectively. These two genes overlap by 8 bases with repB situated upstream. The replicon also transcribes an antisense RNA (RNAI) that inhibits the expression of repA and repB. The ribosome-binding sequence (RBS) of repA is likely to be hidden in a stem-loop structure, inhibiting the translation of repA. Furthermore, translation of repB is likely to disrupt the stem-loop structure, which is one of the criteria allowing the translation of repA to begin. A mutagenesis study revealed that a sequence (5'-GCACGGG-3') located 111 nt upstream from repA is crucial; mutation of this sequence prevented the translation of repA. Additionally, this region and the stem-loop structure containing the RBS of repA may form an RNA pseudoknot. Results in this study demonstrate that a mechanism similar to that regulating plasmid replication in the IncB, IncIalpha and IncL/M groups also regulates pSW800 replication. PMID:11577155

  5. Naked singularities as particle accelerators. II

    SciTech Connect

    Patil, Mandar; Joshi, Pankaj S.; Malafarina, Daniele

    2011-03-15

    We generalize here our earlier results on particle acceleration by naked singularities. We showed recently [M. Patil and P. S. Joshi, Phys. Rev. D 82, 104049 (2010).] that the naked singularities that form due to the gravitational collapse of massive stars provide a suitable environment where particles could get accelerated and collide at arbitrarily high center-of-mass energies. However, we focused there only on the spherically symmetric gravitational collapse models, which were also assumed to be self-similar. In this paper, we broaden and generalize the result to all gravitational collapse models leading to the formation of a naked singularity as the final state of collapse, evolving from a regular initial data, without making any prior restrictive assumptions about the spacetime symmetries such as above. We show that, when the particles interact and collide near the Cauchy horizon, the energy of collision in the center-of-mass frame will be arbitrarily high, thus offering a window to the Planck scale physics. We also consider the issue of various possible physical mechanisms of generation of such very high-energy particles from the vicinity of naked singularity. We then construct a model of gravitational collapse to a timelike naked singularity to demonstrate the working of these ideas, where the pressure is allowed to be negative, but the energy conditions are respected. We show that a finite amount of mass-energy density has to be necessarily radiated away from the vicinity of the naked singularity as the collapse evolves. Therefore, the nature of naked singularities, both at the classical and quantum level, could play an important role in the process of particle acceleration, explaining the occurrence of highly energetic outgoing particles in the vicinity of the Cauchy horizon that participate in extreme high-energy collisions.

  6. Seroepidemiology of Polioviruses among University Students in Northern Italy

    PubMed Central

    Baldo, Vincenzo; Cocchio, Silvia; Lazzari, Roberta; Saracino, Elena; Bertoncello, Chiara; Buja, Alessandra; Trevisan, Andrea

    2012-01-01

    The widespread use of poliovirus vaccination schemes has led to a marked decline in the incidence of paralytic poliomyelitis worldwide, but wild poliovirus is still endemic in some developing countries, and in 2009 a total of 23 countries reported at least 1 case of poliomyelitis caused by wild-strain polio viruses. A serological survey was thus conducted on the immunological status against polioviruses of 318 young adults, classified by their country of origin. Immunity to poliomyelitis was assessed by neutralizing antibody titration in tissues cultured on microplates. The rate of seronegativity (≤1:8) in the study population was 26.7% for poliovirus type 1, 7.2% for type 2, and 22.6% for type 3. In our sample of 318 individuals, 219 (68.9%) were Italian and 99 (31.1%) were from outside the European Union (EU). The proportion of cases found seropositive to polioviruses 1 and 3 decreased significantly with older age; this age-related decrease was more evident in the Italian group than among the non-EU subjects. Any risk of the wild virus recurring and causing paralytic poliomyelitis must be prevented, keeping Europe polio free by means of appropriate immunological protection, until polio has been conclusively eradicated all over the world. Judging from our findings, it may be worth considering administering a fifth dose of polio vaccine to adolescents. PMID:22739695

  7. Seroepidemiology of polioviruses among university students in northern Italy.

    PubMed

    Baldo, Vincenzo; Baldovin, Tatjana; Cocchio, Silvia; Lazzari, Roberta; Saracino, Elena; Bertoncello, Chiara; Buja, Alessandra; Trevisan, Andrea

    2012-08-01

    The widespread use of poliovirus vaccination schemes has led to a marked decline in the incidence of paralytic poliomyelitis worldwide, but wild poliovirus is still endemic in some developing countries, and in 2009 a total of 23 countries reported at least 1 case of poliomyelitis caused by wild-strain polio viruses. A serological survey was thus conducted on the immunological status against polioviruses of 318 young adults, classified by their country of origin. Immunity to poliomyelitis was assessed by neutralizing antibody titration in tissues cultured on microplates. The rate of seronegativity (≤ 1:8) in the study population was 26.7% for poliovirus type 1, 7.2% for type 2, and 22.6% for type 3. In our sample of 318 individuals, 219 (68.9%) were Italian and 99 (31.1%) were from outside the European Union (EU). The proportion of cases found seropositive to polioviruses 1 and 3 decreased significantly with older age; this age-related decrease was more evident in the Italian group than among the non-EU subjects. Any risk of the wild virus recurring and causing paralytic poliomyelitis must be prevented, keeping Europe polio free by means of appropriate immunological protection, until polio has been conclusively eradicated all over the world. Judging from our findings, it may be worth considering administering a fifth dose of polio vaccine to adolescents. PMID:22739695

  8. 4D Visualization of replication foci in mammalian cells corresponding to individual replicons

    PubMed Central

    Chagin, V. O.; Casas-Delucchi, C. S.; Reinhart, M.; Schermelleh, L.; Markaki, Y.; Maiser, A.; Bolius, J. J.; Bensimon, A.; Fillies, M.; Domaing, P.; Rozanov, Y. M.; Leonhardt, H.; Cardoso, M. C.

    2016-01-01

    Since the pioneering proposal of the replicon model of DNA replication 50 years ago, the predicted replicons have not been identified and quantified at the cellular level. Here, we combine conventional and super-resolution microscopy of replication sites in live and fixed cells with computational image analysis. We complement these data with genome size measurements, comprehensive analysis of S-phase dynamics and quantification of replication fork speed and replicon size in human and mouse cells. These multidimensional analyses demonstrate that replication foci (RFi) in three-dimensional (3D) preserved somatic mammalian cells can be optically resolved down to single replicons throughout S-phase. This challenges the conventional interpretation of nuclear RFi as replication factories, that is, the complex entities that process multiple clustered replicons. Accordingly, 3D genome organization and duplication can be now followed within the chromatin context at the level of individual replicons. PMID:27052570

  9. 4D Visualization of replication foci in mammalian cells corresponding to individual replicons.

    PubMed

    Chagin, V O; Casas-Delucchi, C S; Reinhart, M; Schermelleh, L; Markaki, Y; Maiser, A; Bolius, J J; Bensimon, A; Fillies, M; Domaing, P; Rozanov, Y M; Leonhardt, H; Cardoso, M C

    2016-01-01

    Since the pioneering proposal of the replicon model of DNA replication 50 years ago, the predicted replicons have not been identified and quantified at the cellular level. Here, we combine conventional and super-resolution microscopy of replication sites in live and fixed cells with computational image analysis. We complement these data with genome size measurements, comprehensive analysis of S-phase dynamics and quantification of replication fork speed and replicon size in human and mouse cells. These multidimensional analyses demonstrate that replication foci (RFi) in three-dimensional (3D) preserved somatic mammalian cells can be optically resolved down to single replicons throughout S-phase. This challenges the conventional interpretation of nuclear RFi as replication factories, that is, the complex entities that process multiple clustered replicons. Accordingly, 3D genome organization and duplication can be now followed within the chromatin context at the level of individual replicons. PMID:27052570

  10. Gravitational lensing by rotating naked singularities

    SciTech Connect

    Gyulchev, Galin N.; Yazadjiev, Stoytcho S.

    2008-10-15

    We model massive compact objects in galactic nuclei as stationary, axially symmetric naked singularities in the Einstein-massless scalar field theory and study the resulting gravitational lensing. In the weak deflection limit we study analytically the position of the two weak field images, the corresponding signed and absolute magnifications as well as the centroid up to post-Newtonian order. We show that there are static post-Newtonian corrections to the signed magnification and their sum as well as to the critical curves, which are functions of the scalar charge. The shift of the critical curves as a function of the lens angular momentum is found, and it is shown that they decrease slightly for the weakly naked and vastly for the strongly naked singularities with the increase of the scalar charge. The pointlike caustics drift away from the optical axis and do not depend on the scalar charge. In the strong deflection limit approximation, we compute numerically the position of the relativistic images and their separability for weakly naked singularities. All of the lensing quantities are compared to particular cases as Schwarzschild and Kerr black holes as well as Janis-Newman-Winicour naked singularities.

  11. Cellular origin and ultrastructure of membranes induced during poliovirus infection.

    PubMed Central

    Schlegel, A; Giddings, T H; Ladinsky, M S; Kirkegaard, K

    1996-01-01

    Poliovirus RNA replicative complexes are associated with cytoplasmic membranous structures that accumulate during viral infection. These membranes were immunoisolated by using a monoclonal antibody against the viral nonstructural protein 2C. Biochemical analysis of the isolated membranes revealed that several organelles of the host cell (lysosomes, trans-Golgi stack and trans-Golgi network, and endoplasmic reticulum) contributed to the virus-induced membranous structures. Electron microscopy of infected cells preserved by high-pressure freezing revealed that the virus-induced membranes contain double lipid bilayers that surround apparently cytosolic material. Immunolabeling experiments showed that poliovirus proteins 2C and 3D were localized to the same membranes as the cellular markers tested. The morphological and biochemical data are consistent with the hypothesis that autophagy or a similar host process is involved in the formation of the poliovirus-induced membranes. PMID:8794292

  12. Poliovirus protein 2C has ATPase and GTPase activities.

    PubMed

    Rodríguez, P L; Carrasco, L

    1993-04-15

    Poliovirus protein 2C belongs to an expanding group of proteins containing a nucleotide binding motif in their sequence. We present evidence that poliovirus 2C has nucleoside triphosphatase (NTPase) activity and binds to RNA. Poliovirus 2C was expressed in Escherichia coli cells as a fusion protein with the maltose binding protein (MBP). The fusion protein MBP-2C is efficiently cut by protease Xa within the 2C region. Thus, the fusion protein as such was used to assay for the putative activities of poliovirus 2C. Deletion mutants were constructed which lacked different portions of the 2C carboxyl terminus: mutant 2C delta 1 lacked the last 169 amino acids, whereas mutant 2C delta 2 had the last 74 amino acids deleted. The fusion proteins MBP-2C, MBP-2BC, and the mutant MBP-2C delta 2 that contained the first 255 amino acids of 2C had NTPase activity. Both ATPase and GTPase activities are inhibited by antibodies directed against the MBP-2C protein. Analysis of the ability of the different proteins to bind to labeled RNA indicates that MBP-2C and MBP-2BC form a complex, whereas none of the mutants interacted with RNA, indicating that the RNA binding domain lies beyond amino acid 255. None of the fusion proteins had detectable helicase activity. We suggest that poliovirus protein 2C shows similarities to the GTPases group involved in vesicular traffic and transports the viral RNA replication complexes. These results provide the first experimental evidence that poliovirus protein 2C is an NTPase and that this protein has affinity for nucleic acids. PMID:8385138

  13. Annual report of the Australian National Poliovirus Reference Laboratory, 2008.

    PubMed

    Roberts, Jason A; Grant, Kristina A; Yoon, Yeon Kyung; Polychronopoulos, Sophie; Ibrahim, Aishah; Thorley, Bruce R

    2009-09-01

    The Australian National Poliovirus Reference Laboratory (NPRL) is accredited by the World Health Organization (WHO) for the testing of stool specimens from cases of acute flaccid paralysis (AFP), a major clinical presentation of poliovirus infection. The NPRL, in collaboration with the Australian Paediatric Surveillance Unit, co-ordinates surveillance for cases of AFP in children in Australia, according to criteria recommended by the WHO. Clinical specimens are referred from AFP cases in children and suspected case of poliomyelitis in persons of any age. The WHO AFP surveillance performance indicator for a polio-free country such as Australia, is 1 non-polio AFP case per 100,000 children less than 15 years of age. In 2008, the Polio Expert Committee (PEC) classified 62 cases as non-polio AFP, or 1.51 non-polio AFP cases per 100,000 children aged less than 15 years. Poliovirus infection is confirmed by virus culture of stool specimens from AFP cases as other conditions that present with acute paralysis can mimic polio. While no poliovirus was reported in Australia from any source in 2008, the non-polio enteroviruses echovirus 25, coxsackievirus B2 and echovirus 11 were isolated from stool specimens of AFP cases. The last report of a wild poliovirus in Australia was due to an importation from Pakistan in 2007. With 4 countries remaining endemic for poliomyelitis--Afghanistan, India, Nigeria and Pakistan--and more than 1,600 confirmed cases of wild poliovirus infection in 18 countries in 2008, Australia continues to be at risk of further importation events. PMID:20043599

  14. A PCR-based protocol for generating West Nile virus replicons.

    PubMed

    Maeda, Junko; Takagi, Hirotaka; Hashimoto, Shingo; Kurane, Ichiro; Maeda, Akihiko

    2008-03-01

    A new protocol for the generation of West Nile virus (WNV) replicons was developed. Fragmented cDNAs that covered the entire WNV RNA sequence, except the sequence corresponding to nucleotides 190-2379, were amplified separately by polymerase chain reactions (PCRs) using primer set franking with overlapping sequences of 40-50 bp at the 5'- and the 3'-ends of each fragment. All amplified fragments were mixed together and annealed to each other at the overlapping sequences. The annealed-DNA fragments were elongated by DNA polymerase and amplified by short-cycle PCRs to generate full-sized WNV replicon cDNAs. The WNV replicons were transcribed in vitro using the replicon cDNAs as templates. When the in vitro-transcribed replicon was introduced into mammalian cells, the viral envelope protein and viral positive- and negative-strand RNAs were detected in the replicon-transfected cells. It is noteworthy that the synthesis of the replicon cDNAs and the replicons took just 1 week, and that the use of a high-fidelity DNA polymerase afforded stability to the sequence of the synthetic replicon. PMID:18242719

  15. Defective Interfering Particles of Poliovirus I. Isolation and Physical Properties

    PubMed Central

    Cole, Charles N.; Smoler, Donna; Wimmer, Eckard; Baltimore, David

    1971-01-01

    A class of defective interfering (DI) poliovirus particles has been identified. The first was found as a contaminant of a viral stock; others have been isolated by serial passage at a high multiplicity of infection. The DI particles are less dense than standard virus and sediment more slowly. Their ribonucleic acid (RNA) sediments more slowly than standard RNA and has a higher electrophoretic mobility. Competition hybridization experiments with double-stranded viral RNA indicate that DI RNA is 80 to 90% of the length of standard RNA. The proteins of DI particles are indistinguishable from those of standard poliovirus. PMID:4329564

  16. Modification of RNA polymerase IIO subspecies after poliovirus infection.

    PubMed Central

    Rangel, L M; Fernandez-Tomas, C; Dahmus, M E; Gariglio, P

    1987-01-01

    Infection of HeLa cells with poliovirus results in a shutdown of host transcription. In an effort to understand the mechanism(s) that underlies this process, we analyzed the distribution of RNA polymerase IIO before and after viral infection. Analysis of free and chromatin-bound enzyme indicated that there is a significant reduction in RNA polymerase IIO following infection. This observation, together with increasing evidence that transcription is catalyzed by RNA polymerase IIO, supports the hypothesis that poliovirus-induced inhibition of host transcription occurs at the level of RNA chain initiation and involves the direct modification of RNA polymerase II. Images PMID:3029396

  17. Class of supersymmetric solitons with naked singularities

    SciTech Connect

    Cvetic, M.; Youm, D. )

    1995-02-15

    We study vacuum domain walls in a class of four-dimensional [ital N]=1 supergravity theories where along with the matter field, forming the wall, there is more than one dilaton,'' each respecting SU(1,1) symmetry in their subsector. We find [ital supersymmetric] (planar, static) walls, interpolating between a Minkowski vacuum and a new class of supersymmetric vacua which have a naked (planar) singularity. Although such walls correspond to idealized configurations, i.e., they correspond to planar configurations of infinite extent, they provide the first example of supersymmetric classical solitons with naked singularities.

  18. Multiple Poliovirus Proteins Repress Cytoplasmic RNA Granules

    PubMed Central

    Dougherty, Jonathan D.; Tsai, Wei-Chih; Lloyd, Richard E.

    2015-01-01

    We have previously shown that poliovirus (PV) infection induces stress granule (SG) formation early in infection and then inhibits the formation of SG and disperses processing bodies (PBs) by the mid-phase of infection. Loss of SG was linked to cleavage of G3BP1 by viral 3C proteinase (3Cpro), however dispersal of PBs was not strongly linked to cleavage of specific factors by viral proteinases, suggesting other viral proteins may play roles in inhibition of SG or PB formation. Here we have screened all viral proteins for roles in inducing or inhibiting the formation of RNA granules by creating fusions with mCherry and expressing them individually in cells. Expression of viral proteins separately revealed that the capsid region P1, 2Apro, 3A, 3Cpro, the protease precursor 3CD and 3D polymerase all affect RNA granules to varying extents, whereas 2BC does not. 2Apro, which cleaves eIF4GI, induced SGs as expected, and entered novel foci containing the SG nucleating protein G3BP1. Of the two forms of G3BP, only G3BP1 is cleaved by a virus proteinase, 3Cpro, whereas G3BP2 is not cleaved by 3Cpro or 2Apro. Surprisingly, 3CD, which contains proteinase activity, differentially repressed PBs but not SGs. Further, both 2Apro and 3Cpro expression dispersed PBs, however molecular targets were different since PB dispersal due to 2Apro and heat shock protein (Hsp)90 inhibition but not 3Cpro, could be rescued by application of oxidative stress to cells. The data indicate that PV repression of SGs and PBs is multifactorial, though protease function is dominant. PMID:26610553

  19. Modeling population immunity to support efforts to end the transmission of live polioviruses.

    PubMed

    Thompson, Kimberly M; Pallansch, Mark A; Tebbens, Radboud J Duintjer; Wassilak, Steve G; Cochi, Stephen L

    2013-04-01

    Eradication of wild poliovirus (WPV) types 1 and 3, prevention and cessation of circulating vaccine-derived polioviruses, and achievement and maintenance of a world free of paralytic polio cases requires active risk management by focusing on population immunity and coordinated cessation of oral poliovirus vaccine (OPV). We suggest the need for a complementary and different conceptual approach to achieve eradication compared to the current case-based approach using surveillance for acute flaccid paralysis (AFP) to identify symptomatic poliovirus infections. Specifically, we describe a modeling approach to characterize overall population immunity to poliovirus transmission. The approach deals with the realities that exposure to live polioviruses (e.g., WPV, OPV) and/or vaccination with inactivated poliovirus vaccine provides protection from paralytic polio (i.e., disease), but does not eliminate the potential for reinfection or asymptomatic participation in poliovirus transmission, which may increase with time because of waning immunity. The AFP surveillance system provides evidence of symptomatic poliovirus infections detected, which indicate immunity gaps after outbreaks occur, and this system represents an appropriate focus for controlling disease outbreaks. We describe a conceptual dynamic model to characterize population immunity to poliovirus transmission that helps identify risks created by immunity gaps before outbreaks occur, which provides an opportunity for national and global policymakers to manage the risk of poliovirus and prevent outbreaks before they occur. We suggest that dynamically modeling risk represents an essential tool as the number of cases approaches zero. PMID:22985171

  20. Poliovirus receptor CD155–targeted oncolysis of glioma1

    PubMed Central

    Merrill, Melinda K.; Bernhardt, Guenter; Sampson, John H.; Wikstrand, Carol J.; Bigner, Darell D.; Gromeier, Matthias

    2004-01-01

    Cell adhesion molecules of the immunoglobulin superfamily are aberrantly expressed in malignant glioma. Amongst these, the human poliovirus receptor CD155 provides a molecular target for therapeutic intervention with oncolytic poliovirus recombinants. Poliovirus has been genetically modified through insertion of regulatory sequences derived from human rhinovirus type 2 to selectively replicate within and destroy cancerous cells. Efficacious oncolysis mediated by poliovirus derivatives depends on the presence of CD155 in targeted tumors. To prepare oncolytic polioviruses for clinical application, we have developed a series of assays in high-grade malignant glioma (HGL) to characterize CD155 expression levels and susceptibility to oncolytic poliovirus recombinants. Analysis of 6 HGL cases indicates that CD155 is expressed in these tumors and in primary cell lines derived from these tumors. Upregulation of the molecular target CD155 rendered explant cultures of all studied tumors highly susceptible to a prototype oncolytic poliovirus recombinant. Our observations support the clinical application of such agents against HGL. PMID:15279713

  1. Phosphorylation of eIF2α is responsible for the failure of the picornavirus internal ribosome entry site to direct translation from Sindbis virus replicons.

    PubMed

    Sanz, Miguel Angel; Redondo, Natalia; García-Moreno, Manuel; Carrasco, Luis

    2013-04-01

    Translation directed by the poliovirus (PV) or encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) is very inefficient when expressed from Sindbis virus (SV) replicons. This inhibition can be rescued by co-expression of PV 2A protease (2A(pro)). Inhibition correlates with the extensive phosphorylation of eukaryotic initiation factor (eIF) 2α induced by SV replication. Confirmation that PV or EMCV IRES-driven translation can function when eIF2α is not phosphorylated was obtained in dsRNA-activated protein kinase knockout mouse embryonic fibroblasts (PKR(-/-) MEFs), where SV replication cannot induce eIF2α phosphorylation, and in variant S51A MEFs that express an unphosphorylatable eIF2α. In these cells, PV or EMCV IRES-dependent translation operated more efficiently than in wild-type MEFs. However, this translation was potently blocked when eIF2α was phosphorylated by the addition of thapsigargin to PKR(-/-) MEFs. In addition, when wild-type eIF2α was expressed in S51A MEFs or PKR was expressed in PKR(-/-) MEFs, PV IRES-dependent translation decreased. In both cases, the decrease in PV IRES-dependent translation correlated with the phosphorylation of eIF2α. Notably, PV 2A(pro) expression rescued PV IRES-driven translation in thapsigargin-treated PKR(-/-) MEFs. Taken together, these results demonstrated that PV IRES-driven translation can take place from SV replicons if eIF2α remains unphosphorylated. Remarkably, PV IRES-dependent translation was fully functional in this system when PV 2A(pro) was present, even if eIF2α was phosphorylated. PMID:23255624

  2. Polio eradication. Efficacy of inactivated poliovirus vaccine in India.

    PubMed

    Jafari, Hamid; Deshpande, Jagadish M; Sutter, Roland W; Bahl, Sunil; Verma, Harish; Ahmad, Mohammad; Kunwar, Abhishek; Vishwakarma, Rakesh; Agarwal, Ashutosh; Jain, Shilpi; Estivariz, Concepcion; Sethi, Raman; Molodecky, Natalie A; Grassly, Nicholas C; Pallansch, Mark A; Chatterjee, Arani; Aylward, R Bruce

    2014-08-22

    Inactivated poliovirus vaccine (IPV) is efficacious against paralytic disease, but its effect on mucosal immunity is debated. We assessed the efficacy of IPV in boosting mucosal immunity. Participants received IPV, bivalent 1 and 3 oral poliovirus vaccine (bOPV), or no vaccine. A bOPV challenge was administered 4 weeks later, and excretion was assessed 3, 7, and 14 days later. Nine hundred and fifty-four participants completed the study. Any fecal shedding of poliovirus type 1 was 8.8, 9.1, and 13.5% in the IPV group and 14.4, 24.1, and 52.4% in the control group by 6- to 11-month, 5-year, and 10-year groups, respectively (IPV versus control: Fisher's exact test P < 0.001). IPV reduced excretion for poliovirus types 1 and 3 between 38.9 and 74.2% and 52.8 and 75.7%, respectively. Thus, IPV in OPV-vaccinated individuals boosts intestinal mucosal immunity. PMID:25146288

  3. 21 CFR 866.3405 - Poliovirus serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Poliovirus serological reagents. 866.3405 Section 866.3405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3405...

  4. 21 CFR 866.3405 - Poliovirus serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Poliovirus serological reagents. 866.3405 Section 866.3405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3405...

  5. 21 CFR 866.3405 - Poliovirus serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Poliovirus serological reagents. 866.3405 Section 866.3405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3405...

  6. 21 CFR 866.3405 - Poliovirus serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Poliovirus serological reagents. 866.3405 Section 866.3405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3405...

  7. 21 CFR 866.3405 - Poliovirus serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Poliovirus serological reagents. 866.3405 Section 866.3405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3405...

  8. POLIOVIRUS TYPE 1: NEUTRALIZATION BY PAPAIN-DIGESTED ANTIBODIES.

    PubMed

    VOGT, A; KOPP, R; MAASS, G; REICH, L

    1964-09-25

    Papain-digested rabbit antibody (Porter's fractions I and II) can neutralize poliovirus. Neutralizing capacity after digestion ranged from 35 to 45 percent of that of the undigested antibody. No definite dissociation of the antibody fragments from the virus was observed after the reaction mixture had been diluted in a neutral medium. PMID:14175107

  9. EVIDENCE FOR THE CHROMOSOMAL REPLICONS AS UNITS OF SISTER CHROMATID EXCHANGES

    EPA Science Inventory

    Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the FCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cycloph...

  10. Mucosal and systemic adjuvant activity of alphavirus replicon particles

    NASA Astrophysics Data System (ADS)

    Thompson, Joseph M.; Whitmore, Alan C.; Konopka, Jennifer L.; Collier, Martha L.; Richmond, Erin M. B.; Davis, Nancy L.; Staats, Herman F.; Johnston, Robert E.

    2006-03-01

    Vaccination represents the most effective control measure in the fight against infectious diseases. Local mucosal immune responses are critical for protection from, and resolution of, infection by numerous mucosal pathogens. Antigen processing across mucosal surfaces is the natural route by which mucosal immunity is generated, as peripheral antigen delivery typically fails to induce mucosal immune responses. However, we demonstrate in this article that mucosal immune responses are evident at multiple mucosal surfaces after parenteral delivery of Venezuelan equine encephalitis virus replicon particles (VRP). Moreover, coinoculation of null VRP (not expressing any transgene) with inactivated influenza virions, or ovalbumin, resulted in a significant increase in antigen-specific systemic IgG and fecal IgA antibodies, compared with antigen alone. Pretreatment of VRP with UV light largely abrogated this adjuvant effect. These results demonstrate that alphavirus replicon particles possess intrinsic systemic and mucosal adjuvant activity and suggest that VRP RNA replication is the trigger for this activity. We feel that these observations and the continued experimentation they stimulate will ultimately define the specific components of an alternative pathway for the induction of mucosal immunity, and if the activity is evident in humans, will enable new possibilities for safe and inexpensive subunit and inactivated vaccines. vaccine vector | Venezuelan equine encephalitis virus | viral immunology | RNA virus

  11. Annual report of the Australian National Poliovirus Reference Laboratory, 2009.

    PubMed

    Roberts, Jason A; Hobday, Linda; Polychronopoulos, Sophie; Ibrahim, Aishah; Thorley, Bruce R

    2010-09-01

    The Australian National Poliovirus Reference Laboratory (NPRL) is accredited by the World Health Organization (WHO) for the testing of faecal specimens from acute flaccid paralysis (AFP) cases and operates as a regional poliovirus reference laboratory for the Western Pacific Region. The NPRL, in collaboration with the Australian Paediatric Surveillance Unit, co-ordinates surveillance for cases of AFP in children in Australia, according to criteria recommended by the WHO. Specimens are referred from AFP cases in children and suspected cases of poliomyelitis in persons of any age. The WHO AFP surveillance performance indicator is 1 non-polio AFP case per 100,000 children less than 15 years of age. In 2009, the Polio Expert Committee classified 48 cases as non-polio AFP, a rate of 1.17 cases per 100,000 children less than 15 years of age. An additional WHO AFP surveillance performance indicator is that more than 80% of notified AFP cases have 2 faecal samples collected 24 hours apart and within 14 days of onset of paralysis. Adequate faecal samples were received from 16 (33.3%) of the 48 classified cases. A poliovirus was referred via the Enterovirus Reference Laboratory Network of Australia from a non-AFP case and was determined to be Sabin-like. This case most likely represents an importation event, the source of which was not identified, as Australia ceased using Sabin oral polio vaccine in 2005. The last report of a wild poliovirus importation in Australia was from Pakistan in 2007. In 2009, 1,604 wild poliovirus cases were reported in 23 countries with Afghanistan, India, Nigeria and Pakistan remaining endemic for poliomyelitis. PMID:21090182

  12. Gravitational radiation from a cylindrical naked singularity

    SciTech Connect

    Nakao, Ken-ichi; Morisawa, Yoshiyuki

    2005-06-15

    We construct an approximate solution which describes the gravitational emission from a naked singularity formed by the gravitational collapse of a cylindrical thick shell composed of dust. The assumed situation is that the collapsing speed of the dust is very large. In this situation, the metric variables are obtained approximately by a kind of linear perturbation analysis in the background Morgan solution which describes the motion of cylindrical null dust. The most important problem in this study is what boundary conditions for metric and matter variables should be imposed at the naked singularity. We find a boundary condition that all the metric and matter variables are everywhere finite at least up to the first order approximation. This implies that the spacetime singularity formed by this high-speed dust collapse is very similar to that formed by the null dust and the final singularity will be a conical one. Weyl curvature is completely released from the collapsed dust.

  13. National choices related to inactivated poliovirus vaccine, innovation and the endgame of global polio eradication.

    PubMed

    Thompson, Kimberly M; Duintjer Tebbens, Radboud J

    2014-02-01

    Achieving the goal of a world free of poliomyelitis still requires significant effort. Although polio immunization represents a mature area, the polio endgame will require new tools and strategies, particularly as national and global health leaders coordinate the cessation of all three serotypes of oral poliovirus vaccine and increasingly adopt inactivated poliovirus vaccine (IPV). Poliovirus epidemiology and the global options for managing polioviruses continue to evolve, along with our understanding and appreciation of the resources needed and the risks that require management. Based on insights from modeling, we offer some perspective on the current status of plans and opportunities to achieve and maintain a world free of wild polioviruses and to successfully implement oral poliovirus vaccine cessation. IPV costs and potential wastage will represent an important consideration for national policy makers. Innovations may reduce future IPV costs, but the world urgently needs lower-cost IPV options. PMID:24308581

  14. NAKE'MUU: VILLAGE ON THE EDGE

    SciTech Connect

    B. VIERRA; ET AL

    2001-02-01

    Pursuant to federal requirements under the National Environmental Policy Act, in 1996 the Department of Energy (DOE) completed an environmental impact statement, record of decision, and mitigation action plan (MAP) for the Dual Axis Radiographic Hydrodynamic Test (DARHT) Facility. DARHT is a new explosives testing facility located at TA-15, Los Alamos National Laboratory (LANL). As part of the implementation of the MAP, the ESH-20 Cultural Resources Team at LANL is conducting a long-term monitoring program at the ancestral pueblo site of Nake'muu (LA 12655). DOE considers the monitoring program to be an appropriate and necessary mitigation for the potential operational impacts associated with the DARHT Facility. This chapter presents the preliminary findings of the monitoring program from 1997 through 2000. The Nake'muu site monitoring program was initiated by ESH-20 and the Mesa Verde Architectural Team, National Park Service (NPS) in 1997. Archaeologists from LANL and NPS have completed the detailed recording of all the walls at Nake'muu and the final site evaluation plan was finished in 1998 (Nordby et al. 1998). The plan describes the methods used for site monitoring, continued site revisits, and updates on wall condition by LANL archaeologists. The primary objective of the monitoring program is to identify and evaluate the long-term effects of the ambient environment and DARHT Facility operations on the architecture at Nake'muu. Is the dynamic-testing program affecting the site, and if so, to what degree? What are the short-term and long-term implications of facility operations?

  15. Radiation effects on DNA synthesis in a defined chromosomal replicon

    SciTech Connect

    Larner, J.M.; Lee, H.; Hamlin, J.L. )

    1994-03-01

    It has recently been shown that the tumor suppressor p53 mediates a signal transduction pathway that responds to DNA damage by arresting cells in the late G[sub 1] period of the cell cycle. However, the operation of this pathway alone cannot explain the 50% reduction in the rate of DNA synthesis that occurs within 30 min of irradiation of an asynchronous cell population. The authors are using the amplified dihydrofolate reductase (DHFR) domain in the methotrexate-resistance CHO cell line, CHOC 400, as a model replicon in which to study this acute radiation effect. They first show that the CHOC-400 cell line retains the classical acute-phase response but does not display the late G[sub 1] arrest that characterizes the p53-mediated checkpoint. Using a two-dimensional gel replicon-mapping method, they then show that when asynchronous cultures are irradiated with 900 cGy, initiation in the DHFR locus is completely inhibited within 30 min and does not resume for 3 to 4 h. Since initiation in this locus occurs throughout the first 2 h of the S period, this result implies the existence of a p53-independent S-phase damage-sensing pathway that functions at the level of individual origins. Results obtained with the replication inhibitor mimosine define a position near the G[sub 1]/S boundary beyond which cells are unable to prevent initiation at early-firing origins in response to irradiation. This is the first direct demonstration at a defined chromosomal origin that radiation quantitatively down-regulates initiation. 42 refs., 9 figs.

  16. Chlorine resistance of poliovirus isolants recovered from drinking water.

    PubMed Central

    Shaffer, P T; Metcalf, T G; Sproul, O J

    1980-01-01

    Poliovirus 1 isolants were recovered from finished drinking water produced by a modern, well-operated water treatment plant. These waters contained free chlorine residuals in excess of 1 mg/liter. The chlorine inactivation of purified high-titer preparations of two such isolants was compared with the inactivation behavior of two stock strains of poliovirus 1, LSc and Mahoney. The surviving fraction of virus derived from the two natural isolants was shown to be orders of magnitude greater than that of the standard strains. These results raise the question whether indirect drinking water standards based on free chlorine residuals are adequate public health measures, or whether direct standards based on virus determinations might be necessary. Images PMID:6257162

  17. INFECTIVITY OF HISTONE-POLIOVIRUS RIBONUCLEIC ACID PREPARATIONS.

    PubMed

    LUDWIG, E H; SMULL, C E

    1963-06-01

    Ludwig, Ernest H. (The Pennsylvania State University, University Park) and Christine E. Smull. Infectivity of histone-poliovirus ribonucleic acid preparations. J. Bacteriol. 85:1334-1338. 1963.-Some properties of histone-poliovirus ribonucleic acid (RNA) preparations, as relate to infectivity for HeLa cell monolayers, were investigated. The histone-RNA preparations were found to lose their infectivity rapidly at room temperature. They were considerably more stable at ice-bath temperature. Dilution of a histone-RNA preparation in a diluent containing an appropriate amount of histone yielded a plaque count which was proportional to the dilution factor, and which regressed linearly through the point of origin. Dilution of a histone-RNA preparation in a diluent containing no histone resulted in a rapidly decreasing plaque count which was not proportional to the dilution factor. The ability of histone to enhance the infectivity of poliovirus RNA was found to be dependent upon the presence of a low concentration of a monovalent salt in the histone-RNA preparations. Histone-RNA preparations containing approximately isotonic levels of NaCl exhibited a high degree of infectivity. When concentrations of NaCl outside this range were used, a great loss in infectivity of the histone-RNA preparations occurred. The NaCl could be replaced by KCl but not by CaCl(2), MgCl(2), or MgSO(4). PMID:14047226

  18. Surface for Catalysis by Poliovirus RNA-Dependent RNA Polymerase

    PubMed Central

    Wang, Jing; Lyle, John M.; Bullitt, Esther

    2013-01-01

    The poliovirus RNA-dependent RNA polymerase, 3Dpol, replicates the viral genomic RNA on the surface of virus-induced intracellular membranes. Macromolecular assemblies of 3Dpol form linear array of subunits that propagate along a strong protein-protein interaction called interface-I, as was observed in the crystal structure of wild-type poliovirus polymerase. These “filaments” recur with slight modifications in planar sheets and, with additional modifications that accommodate curvature, in helical tubes of the polymerase, by packing filaments together via a second set of interactions. Periodic variations of subunit orientations within 3Dpol tubes give rise to “ghost reflections” in diffraction patterns computed from electron cryomicrographs of helical arrays. The ghost reflections reveal that polymerase tubes are formed by bundles of 4–6 interface-I filaments, which are then connected to the next bundle of filaments with a perturbation of interface interactions between bundles. While enzymatically inactive polymerase is also capable of oligomerization, much thinner tubes are formed that lack interface-I interactions between adjacent subunits, suggesting that long-range allostery produces conformational changes that extend from the active site to the protein-protein interface. Macromolecular assemblies of poliovirus polymerase show repeated use of flexible interface interactions for polymerase lattice formation, suggesting that adaptability of polymerase-polymerase interactions facilitates RNA replication. In addition, the presence of a positively charged groove identified in polymerase arrays may help position and stabilize the RNA template during replication. PMID:23583774

  19. Antimicrobial Susceptibility and Plasmid Replicon Typing of Salmonella enterica serovar Kentucky isolates recovered from Broilers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Salmonella Kentucky has become the predominate serotype recovered from broiler slaughter in the United States and the prevalence of antimicrobial resistance (AMR) has increased dramatically in this serotype. Relationships between AMR, genotype, and plasmid replicon types were characterized for 600 ...

  20. The organization of repeated nucleotide sequences in the replicons of mammalian DNA.

    PubMed Central

    Mattern, M R; Painter, R B

    1977-01-01

    Chinese hamster ovary cells were irradiated with 100-5,000 rads of X-rays and inhibition of the initiation of replicons after irradiation was demonstrated by analyzing nascent DNA sedimented in alkaline sucrose gradients. The renaturation kinetics of DNA synthesized during 60 min of incubation after irradiation was compared with that of DNA synthesized during the 60 min after sham irradiation and with that of parental DNA. Nascent DNA from cells whose replicon initiation was inhibited renatured faster than nascent DNA from control cells in the COt range of repeated nucleotide sequences, suggesting that regions of the replicon not close to origins are enriched in repeated sequences and that regions close to origins are enriched in unique sequences. A class of repeated nucleotide sequences may be involved in the regulation of replicon initiation. PMID:880330

  1. Visibility limit of naked-eye sunspots

    NASA Astrophysics Data System (ADS)

    Keller, H. U.; Friedli, T. K.

    1992-06-01

    Based on observations of 20 observers, a visibility limit for naked-eye sunspot observations has been calculated. A sunspot with a penumbral diameter of at least 41 arcsec and an umbral diameter of at least 15 arcsec can be detected by an average eye. An eyesight test was designed to solve the question whether the penumbral or the umbral diameter is conclusive for the visibility of a sunspot. The measured limit of 19.3 arcsec indicates that a combination of both is the determining factor.

  2. The effects of convective overshooting on naked helium stars

    NASA Astrophysics Data System (ADS)

    Yan, Jing-Zhi; Zhu, Chun-Hua; Wang, Zhao-Jun; Lü, Guo-Liang

    2016-09-01

    Using stellar evolutionary models, we investigate the effects of convective overshooting on naked helium stars. We find that a larger value of overshooting parameter δov results in a larger convective core, which prolongs the lifetimes of naked helium stars on the helium main sequence and leads to higher effective temperatures and luminosities. For naked helium stars with masses lower than about 0.8 M⊙, they hardly become giant stars as a result of a weak burning shell. However, naked helium stars with masses between about 0.8 M⊙ and 1.1 M⊙ can evolve into giant branch phases, and finally become carbon oxygen white dwarfs.

  3. Hepatitis C Virus RNA Elimination and Development of Resistance in Replicon Cells Treated with BMS-790052

    PubMed Central

    Wang, Chunfu; Huang, Haichang; Valera, Lourdes; Sun, Jin-Hua; O'Boyle, Donald R.; Nower, Peter T.; Jia, Lingling; Qiu, Dike; Huang, Xin; Altaf, Aneela; Gao, Min

    2012-01-01

    BMS-790052, a first-in-class hepatitis C virus (HCV) replication complex inhibitor, targeting nonstructural protein 5A (NS5A), displays picomolar to nanomolar potency against genotypes 1 to 5. This exceptional potency translated into robust anti-HCV activity in clinical studies with HCV genotype 1-infected subjects. To date, all BMS-790052-associated resistance mutations have mapped to the N-terminal region of NS5A. To further characterize the antiviral activity of BMS-790052, HCV replicon elimination and colony formation assays were performed. Replicon was cleared from genotype 1a and 1b replicon cells in a time- and dose-dependent manner. Elimination of the genotype 1a replicon required longer treatment durations and higher concentrations of BMS-790052 than those for the genotype1b replicon. Single amino acid substitutions that conferred relatively low levels of resistance were observed at early time points and at low doses. Higher doses and longer treatment durations yielded mutations that conferred greater levels of resistance, including linked amino acid substitutions. Replicon cells that survived inhibitor treatment remained fully sensitivity to pegylated alpha interferon (pegIFN-α) and other HCV inhibitors. Moreover, genotype 1a replicon elimination was markedly enhanced when pegIFN-α and BMS-790052 were combined. Resistant variants observed in this study were very similar to those observed in a multiple ascending dose (MAD) monotherapy trial of BMS-790052, validating replicon elimination studies as a model to predict clinical resistance. Insights gained from the in vitro anti-HCV activity and resistance profiles of BMS-790052 will be used to help guide the clinical development of this novel HCV inhibitor. PMID:22214777

  4. Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

    PubMed Central

    Kaufmann, W K; Boyer, J C; Estabrooks, L L; Wilson, S J

    1991-01-01

    Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes. PMID:1646393

  5. Assessment of cell culture and polymerase chain reaction procedures for the detection of polioviruses in wastewater.

    PubMed Central

    Grabow, W. O.; Botma, K. L.; de Villiers, J. C.; Clay, C. G.; Erasmus, B.

    1999-01-01

    WHO considers that environmental surveillance for wild-type polioviruses is potentially important for surveillance for acute flaccid paralysis as a means of confirming eradication of poliomyelitis. The present study investigated methods for detecting polioviruses in a variety of water environments in South Africa. Most polioviruses were isolated on L20B mouse cells, which, however, were not selective: 16 reoviruses and 8 enteroviruses, apparently animal strains, were also isolated on these cells. Vaccine strains of polioviruses were isolated from surface waters during and shortly after two rounds of mass vaccination of children in an informal settlement where there was no sewerage. The results demonstrated the feasibility of poliovirus surveillance in such settlements. It was also evident that neither poliovirus vaccine strains nor other viruses were likely to interfere significantly with the detection of wild-type polioviruses. Optimal isolation of polioviruses was accomplished by parallel inoculation of L20B mouse cells and at least the PLC/PRF/5 human liver and buffalo green monkey (BGM) kidney cell lines. Analysis of cell cultures using the polymerase chain reaction revealed that 319 test samples contained at least 263 human enteroviruses that failed to produce a cytopathogenic effect. This type of analysis thus significantly increased the sensitivity of enterovirus detection. PMID:10680244

  6. Development and introduction of inactivated poliovirus vaccines derived from Sabin strains in Japan.

    PubMed

    Shimizu, Hiroyuki

    2016-04-01

    During the endgame of global polio eradication, the universal introduction of inactivated poliovirus vaccines is urgently required to reduce the risk of vaccine-associated paralytic poliomyelitis and polio outbreaks due to wild and vaccine-derived polioviruses. In particular, the development of inactivated poliovirus vaccines (IPVs) derived from the attenuated Sabin strains is considered to be a highly favorable option for the production of novel IPV that reduce the risk of facility-acquired transmission of poliovirus to the communities. In Japan, Sabin-derived IPVs (sIPVs) have been developed and introduced for routine immunization in November 2012. They are the first licensed sIPVs in the world. Consequently, trivalent oral poliovirus vaccine was used for polio control in Japan for more than half a century but has now been removed from the list of vaccines licensed for routine immunization. This paper reviews the development, introduction, characterization, and global status of IPV derived from attenuated Sabin strains. PMID:25448090

  7. Evidence of presence of poliovirus genomic sequences in cerebrospinal fluid from patients with postpolio syndrome.

    PubMed

    Leparc-Goffart, I; Julien, J; Fuchs, F; Janatova, I; Aymard, M; Kopecka, H

    1996-08-01

    The postpolio syndrome (PPS) is characterized by new neuromuscular symptoms occurring 30 to 40 years after the acute episode of poliomyelitis paralysis. The presence of the poliovirus RNA genome in the cerebrospinal fluid from 10 patients with PPS and from 23 control patients was sought by using reverse transcription and a PCR specific for polioviruses and/or other enteroviruses. Poliovirus-specific genomic sequences in the 5' untranslated region and in the capsid region (VP1) were detected by reverse transcription PCR in 5 of 10 patients with PPS but in none of the control patients. Sequencing confirmed the presence of mutated poliovirus sequences. This finding suggests persistent viral infection in the central nervous system related to the presence of poliovirus genomes. PMID:8818905

  8. Managing population immunity to reduce or eliminate the risks of circulation following the importation of polioviruses.

    PubMed

    Thompson, Kimberly M; Kalkowska, Dominika A; Duintjer Tebbens, Radboud J

    2015-03-24

    Poliovirus importations into polio-free countries represent a major concern during the final phases of global eradication of wild polioviruses (WPVs). We extend dynamic transmission models to demonstrate the dynamics of population immunity out through 2020 for three countries that only used inactivated poliovirus vaccine (IPV) for routine immunization: the US, Israel, and The Netherlands. For each country, we explore the vulnerability to re-established transmission following an importation for each poliovirus serotype, including the impact of immunization choices following the serotype 1 WPV importation that occurred in 2013 in Israel. As population immunity declines below the threshold required to prevent transmission, countries become at risk for re-established transmission. Although importations represent stochastic events that countries cannot fully control because people cross borders and polioviruses mainly cause asymptomatic infections, countries can ensure that any importations die out. Our results suggest that the general US population will remain above the threshold for transmission through 2020. In contrast, Israel became vulnerable to re-established transmission of importations of live polioviruses by the late 2000s. In Israel, the recent WPV importation and outbreak response use of bivalent oral poliovirus vaccine (bOPV) eliminated the vulnerability to an importation of poliovirus serotypes 1 and 3 for several years, but not serotype 2. The Netherlands experienced a serotype 1 WPV outbreak in 1992-1993 and became vulnerable to re-established transmission in religious communities with low vaccine acceptance around the year 2000, although the general population remains well-protected from widespread transmission. All countries should invest in active management of population immunity to avoid the potential circulation of imported live polioviruses. IPV-using countries may wish to consider prevention opportunities and/or ensure preparedness for response

  9. Naked shell singularities on the brane

    SciTech Connect

    Seahra, Sanjeev S.

    2005-04-15

    By utilizing nonstandard slicings of 5-dimensional Schwarzschild and Schwarzschild-AdS manifolds based on isotropic coordinates, we generate static and spherically-symmetric braneworld spacetimes containing shell-like naked null singularities. For planar slicings, we find that the brane-matter sourcing the solution is a perfect fluid with an exotic equation of state and a pressure singularity where the brane crosses the bulk horizon. From a relativistic point of view, such a singularity is required to maintain matter infinitesimally above the surface of a black hole. From the point of view of the AdS/CFT conjecture, the singular horizon can be seen as one possible quantum correction to a classical black hole geometry. Various generalizations of planar slicings are also considered for a Ricci-flat bulk, and we find that singular horizons and exotic matter distributions are common features.

  10. Airglow events visible to the naked eye

    NASA Technical Reports Server (NTRS)

    Peterson, A. W.

    1979-01-01

    During IR photographic airglow observations covering several years, three naked-eye events have been recorded. Two of these are moving luminous acoustic-gravity-wave groups of some 10-15-km wavelength, which occur near high lunar tide in the atmosphere. The events appear quickly, endure 0.5-1 h, then fade. Visible photos of two events appear enhanced, while little enhancement is present in the IR photos, although the structures are well correlated. If these events are due to OH, it is suggested that some unrecognized mechanism, perhaps a gravity wave interaction, enhances the visible transitions of the OH over the IR transitions. If the events are due to an unrecognized continuum emitter, perhaps NO, its emission must occur at the same height as the OH. Spectra seem to be the only reasonable approach to solving this problem.

  11. Go naked: diapers affect infant walking.

    PubMed

    Cole, Whitney G; Lingeman, Jesse M; Adolph, Karen E

    2012-11-01

    In light of cross-cultural and experimental research highlighting effects of childrearing practices on infant motor skill, we asked whether wearing diapers, a seemingly innocuous childrearing practice, affects infant walking. Diapers introduce bulk between the legs, potentially exacerbating infants' poor balance and wide stance. We show that walking is adversely affected by old-fashioned cloth diapers, and that even modern disposable diapers - habitually worn by most infants in the sample - incur a cost relative to walking naked. Infants displayed less mature gait patterns and more missteps and falls while wearing diapers. Thus, infants' own diapers constitute an ongoing biomechanical perturbation while learning to walk. Furthermore, shifts in diapering practices may have contributed to historical and cross-cultural differences in infant walking. PMID:23106732

  12. A naked ape would have fewer parasites.

    PubMed

    Pagel, Mark; Bodmer, Walter

    2003-08-01

    Unusually among the mammals, humans lack an outer layer of protective fur or hair. We propose the hypothesis that humans evolved hairlessness to reduce parasite loads, especially ectoparasites that may carry disease. We suggest that hairlessness is maintained by these naturally selected benefits and by sexual selection operating on both sexes. Hairlessness is made possible in humans owing to their unique abilities to regulate their environment via fire, shelter and clothing. Clothes and shelters allow a more flexible response to the external environment than a permanent layer of fur and can be changed or cleaned if infested with parasites. Naked molerats, another hairless and non-aquatic mammal species, also inhabit environments in which ectoparasite transmission is expected to be high, but in which temperatures are closely regulated. Our hypothesis explains features of human hairlessness-such as the marked sex difference in body hair, and its retention in the pubic regions-that are not explained by other theories. PMID:12952654

  13. A personal reflection on the replicon theory: from R1 plasmid to replication timing regulation in human cells.

    PubMed

    Masai, Hisao

    2013-11-29

    Fifty years after the Replicon Theory was originally presented, detailed mechanistic insight into prokaryotic replicons has been obtained and rapid progress is being made to elucidate the more complex regulatory mechanisms of replicon regulation in eukaryotic cells. Here, I present my personal perspectives on how studies of model replicons have contributed to our understanding of the basic mechanisms of DNA replication as well as the evolution of replication regulation in human cells. I will also discuss how replication regulation contributes to the stable maintenance of the genome and how disruption of replication regulation leads to human diseases. PMID:23579064

  14. Cross-neutralizing human anti-poliovirus antibodies bind the recognition site for cellular receptor

    PubMed Central

    Chen, Zhaochun; Fischer, Elizabeth R.; Kouiavskaia, Diana; Hansen, Bryan T.; Ludtke, Steven J.; Bidzhieva, Bella; Makiya, Michelle; Agulto, Liane; Purcell, Robert H.; Chumakov, Konstantin

    2013-01-01

    Most structural information about poliovirus interaction with neutralizing antibodies was obtained in the 1980s in studies of mouse monoclonal antibodies. Recently we have isolated a number of human/chimpanzee anti-poliovirus antibodies and demonstrated that one of them, MAb A12, could neutralize polioviruses of both serotypes 1 and 2. This communication presents data on isolation of an additional cross-neutralizing antibody (F12) and identification of a previously unknown epitope on the surface of poliovirus virions. Epitope mapping was performed by sequencing of antibody-resistant mutants and by cryo-EM of complexes of virions with Fab fragments. The results have demonstrated that both cross-neutralizing antibodies bind the site located at the bottom of the canyon surrounding the fivefold axis of symmetry that was previously shown to interact with cellular poliovirus receptor CD155. However, the same antibody binds to serotypes 1 and 2 through different specific interactions. It was also shown to interact with type 3 poliovirus, albeit with about 10-fold lower affinity, insufficient for effective neutralization. Antibody interaction with the binding site of the cellular receptor may explain its broad reactivity and suggest that further screening or antibody engineering could lead to a universal antibody capable of neutralizing all three serotypes of poliovirus. PMID:24277851

  15. Poliovirus Proteins Induce Membrane Association of GTPase ADP-Ribosylation Factor

    PubMed Central

    Belov, George A.; Fogg, Mark H.; Ehrenfeld, Ellie

    2005-01-01

    Poliovirus infection results in the disintegration of intracellular membrane structures and formation of specific vesicles that serve as sites for replication of viral RNA. The mechanism of membrane rearrangement has not been clearly defined. Replication of poliovirus is sensitive to brefeldin A (BFA), a fungal metabolite known to prevent normal function of the ADP-ribosylation factor (ARF) family of small GTPases. During normal membrane trafficking in uninfected cells, ARFs are involved in vesicle formation from different intracellular sites through interaction with numerous regulatory and coat proteins as well as in regulation of phospholipase D activity and cytoskeleton modifications. We demonstrate here that ARFs 3 and 5, but not ARF6, are translocated to membranes in HeLa cell extracts that are engaged in translation of poliovirus RNA. The accumulation of ARFs on membranes correlates with active replication of poliovirus RNA in vitro, whereas ARF translocation to membranes does not occur in the presence of BFA. ARF translocation can be induced independently by synthesis of poliovirus 3A or 3CD proteins, and we describe mutations that abolished this activity. In infected HeLa cells, an ARF1-enhanced green fluorescent protein fusion redistributes from Golgi stacks to the perinuclear region, where poliovirus RNA replication occurs. Taken together, the data suggest an involvement of ARF in poliovirus RNA replication. PMID:15890959

  16. Antimicrobial Susceptibility and Plasmid Replicon Typing of Salmonella enterica Serovar Kentucky Isolates Recovered from Broilers.

    PubMed

    Ladely, Scott R; Meinersmann, Richard J; Ball, Takiyah A; Fedorka-Cray, Paula J

    2016-06-01

    Salmonella Kentucky has become the predominant serovar recovered from broilers slaughtered in the United States, and the prevalence of antimicrobial resistance (AMR) has increased dramatically in this serovar. Relationships between AMR, genotype, and plasmid replicon types were characterized for 600 Salmonella Kentucky isolates recovered from chicken carcasses from 2004 to 2013. Pulsed-field gel electrophoresis cluster analysis revealed 112 unique types sharing 79% similarity. Over half of the isolates studies were assigned to two large clusters (unique restriction patterns) consisting of 190 (A) and 151 (B) isolates. The remaining (n = 259) more diverse isolates (110 unique patterns) shall be designated cluster C for discussion. Clusters A had significantly more (p < 0.05) isolates resistant to streptomycin (68.4%) and tetracycline (91.6%) compared to cluster C (50.6% and 40.9% to streptomycin and tetracycline, respectively) or cluster B, which had the least (p < 0.05) resistance (11.9% and 13.2% to streptomycin and tetracycline, respectively). In addition, there was segregation of plasmid replicon types among clusters. Cluster A had significantly more (p < 0.05) replicon type FIB (90.5%) compared to cluster C (37.1%), which had significantly more compared to cluster B (10.6%). Cluster B had significantly more (p < 0.05) replicon type I1 (87.4%) compared to cluster C (68.7%), which had significantly more (p < 0.05) compared to cluster A (32.6%). Cluster C harbored significantly more (p < 0.05) HI2 replicon type (18.1%) compared to clonal clusters A (1.6%) or B (1.3%). The prevalence of plasmid replicon type A/C did not differ among clusters (A, 0.5%; B, 2.0%; C, 0.4%). Both streptomycin and tetracycline resistance were significantly linked (p < 0.05) to plasmid replicon type FIB. In addition, replicon type HI2 was also significantly linked (p < 0.05) to streptomycin resistance. We conclude that the dramatic increase in

  17. Identification and Manipulation of the Molecular Determinants Influencing Poliovirus Recombination

    PubMed Central

    Runckel, Charles; Westesson, Oscar; Andino, Raul; DeRisi, Joseph L.

    2013-01-01

    The control and prevention of communicable disease is directly impacted by the genetic mutability of the underlying etiological agents. In the case of RNA viruses, genetic recombination may impact public health by facilitating the generation of new viral strains with altered phenotypes and by compromising the genetic stability of live attenuated vaccines. The landscape of homologous recombination within a given RNA viral genome is thought to be influenced by several factors; however, a complete understanding of the genetic determinants of recombination is lacking. Here, we utilize gene synthesis and deep sequencing to create a detailed recombination map of the poliovirus 1 coding region. We identified over 50 thousand breakpoints throughout the genome, and we show the majority of breakpoints to be concentrated in a small number of specific “hotspots,” including those associated with known or predicted RNA secondary structures. Nucleotide base composition was also found to be associated with recombination frequency, suggesting that recombination is modulated across the genome by predictable and alterable motifs. We tested the predictive utility of the nucleotide base composition association by generating an artificial hotspot in the poliovirus genome. Our results imply that modification of these motifs could be extended to whole genome re-designs for the development of recombination-deficient, genetically stable live vaccine strains. PMID:23408891

  18. High resolution identity testing of inactivated poliovirus vaccines

    PubMed Central

    Mee, Edward T.; Minor, Philip D.; Martin, Javier

    2015-01-01

    Background Definitive identification of poliovirus strains in vaccines is essential for quality control, particularly where multiple wild-type and Sabin strains are produced in the same facility. Sequence-based identification provides the ultimate in identity testing and would offer several advantages over serological methods. Methods We employed random RT-PCR and high throughput sequencing to recover full-length genome sequences from monovalent and trivalent poliovirus vaccine products at various stages of the manufacturing process. Results All expected strains were detected in previously characterised products and the method permitted identification of strains comprising as little as 0.1% of sequence reads. Highly similar Mahoney and Sabin 1 strains were readily discriminated on the basis of specific variant positions. Analysis of a product known to contain incorrect strains demonstrated that the method correctly identified the contaminants. Conclusion Random RT-PCR and shotgun sequencing provided high resolution identification of vaccine components. In addition to the recovery of full-length genome sequences, the method could also be easily adapted to the characterisation of minor variant frequencies and distinction of closely related products on the basis of distinguishing consensus and low frequency polymorphisms. PMID:26049003

  19. Poliovirus concentration from tap water with electropositive adsorbent filters.

    PubMed

    Sobsey, M D; Glass, J S

    1980-08-01

    Simple, reliable, and efficient concentration of poliovirus from tap water was obtained with two types of electropositive filter media, one of which is available in the form of a pleated cartridge filter (Virozorb 1MDS). Virus adsorption from tap water between pH 3.5 and 7.5 was more efficient with electropositive filters than with Filterite filters. Elution of adsorbed viruses was more efficient with beef extract in glycine, pH 9.5, than with glycine-NaOH, pH 11.0. In paired comparative studies, electropositive filters, with adsorption at pH 7.5 and no added polyvalent cation salts, gave less variable virus concentration efficiencies than did Filterite filters with adsorption at pH 3.5 plus added MgCl2. Recovery of poliovirus from 1,000-liter tap water volumes was approximately 30% efficient with both Virozorb 1MDS and Filterite pleated cartridge filters, but the former were much simpler to use. The virus adsorption behavior of these filters appears to be related to their surface charge properties, with more electropositive filters giving more efficient virus adsorption from tap water at higher pH levels. PMID:6258472

  20. Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti.

    PubMed

    diCenzo, George C; Checcucci, Alice; Bazzicalupo, Marco; Mengoni, Alessio; Viti, Carlo; Dziewit, Lukasz; Finan, Turlough M; Galardini, Marco; Fondi, Marco

    2016-01-01

    The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes. PMID:27447951

  1. Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti

    PubMed Central

    diCenzo, George C.; Checcucci, Alice; Bazzicalupo, Marco; Mengoni, Alessio; Viti, Carlo; Dziewit, Lukasz; Finan, Turlough M.; Galardini, Marco; Fondi, Marco

    2016-01-01

    The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes. PMID:27447951

  2. A transcriptionally active form of TFIIIC is modified in poliovirus-infected HeLa cells.

    PubMed Central

    Clark, M E; Dasgupta, A

    1990-01-01

    In HeLa cells, RNA polymerase III (pol III)-mediated transcription is severely inhibited by poliovirus infection. This inhibition is due primarily to the reduction in transcriptional activity of the pol III transcription factor TFIIIC in poliovirus-infected cells. However, the specific binding of TFIIIC to the VAI gene B-box sequence, as assayed by DNase I footprinting, is not altered by poliovirus infection. We have used gel retardation analysis to analyze TFIIIC-DNA complexes formed in nuclear extracts prepared from mock- and poliovirus-infected cells. In mock-infected cell extracts, two closely migrating TFIIIC-containing complexes, complexes I and II, were detected in the gel retardation assay. The slower migrating complex, complex I, was absent in poliovirus-infected cell extracts, and an increase occurred in the intensity of the faster-migrating complex (complex II). Also, in poliovirus-infected cell extracts, a new, rapidly migrating complex, complex III, was formed. Complex III may have been the result of limited proteolysis of complex I or II. These changes in TFIIIC-containing complexes in poliovirus-infected cell extracts correlated kinetically with the decrease in TFIIIC transcriptional activity. Complexes I, II, and III were chromatographically separated; only complex I was transcriptionally active and specifically restored pol III transcription when added to poliovirus-infected cell extracts. Acid phosphatase treatment partially converted complex I to complex II but did not affect the binding of complex II or III. Dephosphorylation and limited proteolysis of TFIIIC are discussed as possible mechanisms for the inhibition of pol III-mediated transcription by poliovirus. Images PMID:2204807

  3. Concentration and Purification of Poliovirus by Ultrafiltration and Isopycnic Centrifugation1

    PubMed Central

    Guskey, Louis E.; Wolff, David A.

    1972-01-01

    A method is described by which poliovirus can be rapidly and simply concentrated by the use of a Diaflo XM-50 ultrafilter membrane. Freon-extracted ultrafilter concentrates banded in CsCl provided a 1,724-fold volumetric concentration of poliovirus. During concentration, trypsin-digested cellular material can pass through the ultrafilter membrane, thus providing a versatile means of degrading and eliminating extraneous contaminating proteins. The ultrafilter concentration system is compared with the CsCl cushion system of poliovirus concentration, and both systems are further compared by banding virus and virus capsid material in CsCl by use of isopycnic centrifugation. Images PMID:4341520

  4. Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus

    PubMed Central

    Verma, Harish; Sharma, Prashant; Yang, Jae Seung; Saletti, Giulietta; Ahmad, Mohammad; Bahl, Sunil K.; Wierzba, Thomas F.; Nandy, Ranjan K.; Deshpande, Jagadish M.; Sutter, Roland W.; Czerkinsky, Cecil

    2016-01-01

    Background The “gold standard” for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine. Methods 199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation. Results An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p<0.001). In the IPV group, α4β7+ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4β7+ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4β7+ IgA- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for

  5. Naked singularities in higher dimensional Vaidya space-times

    SciTech Connect

    Ghosh, S. G.; Dadhich, Naresh

    2001-08-15

    We investigate the end state of the gravitational collapse of a null fluid in higher-dimensional space-times. Both naked singularities and black holes are shown to be developing as the final outcome of the collapse. The naked singularity spectrum in a collapsing Vaidya region (4D) gets covered with the increase in dimensions and hence higher dimensions favor a black hole in comparison to a naked singularity. The cosmic censorship conjecture will be fully respected for a space of infinite dimension.

  6. Oral and Inactivated Poliovirus Vaccines in the Newborn: A review

    PubMed Central

    Mateen, Farrah J.; Shinohara, Russell T.; Sutter, Roland W.

    2015-01-01

    Background Oral poliovirus vaccine (OPV) remains the vaccine-of-choice for routine immunization and supplemental immunization activities (SIAs) to eradicate poliomyelitis globally. Recent data from India suggested lowerthanexpected immunogenicity of an OPV birth dose, prompting a review of the immunogenicity of OPV or inactivated poliovirus vaccine (IPV) when administered at birth. Methods We evaluated the seroconversion and reported adverse events among infants given a single birth dose (given ≤7 days of life) of OPV or IPV through a systematic review of published articles and conference abstracts from 1959-2011 in any language found on PubMed, Google Scholar, or reference lists of selected articles. Results 25 articles from 13 countries published between1959 and 2011 documented seroconversion rates in newborns following an OPV dose given within the first seven days of life. There were 10 studies that measured seroconversion rates between 4 and 8 weeks of a single birth dose of TOPV, using an umbilical cord blood draw at the time of birth to establish baseline antibody levels. The percentage of newborns who seroconverted at 8 weeks range 6-42% for poliovirus type 1, 2-63% for type 2, and 1-35% for type 3). For mOPV type 1, seroconversion ranged from 10-76%; mOPV type 3, the range was 12-58%; and for the one study reporting bOPV, it was 20% for type 1 and 7% for type 3. There were four studies of IPV in newborns with a seroconversion rate of 8-100% for serotype 1, 15-100% for serotype 2, and 15-94% for serotype 3, measured at 4-6 weeks of life. No serious adverse events related to newborn OPV or IPV dosing were reported, including no cases of acute flaccid paralysis. Conclusions There is great variability of the immunogenicity of a birth dose of OPV for reasons largely unknown. Our review confirms the utility of a birth dose of OPV, particularly in countries where early induction of polio immunity is imperative. IPV has higher seroconversion rates in newborns and

  7. A naked-eye colorimetric "PCR developer"

    NASA Astrophysics Data System (ADS)

    Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated <10 billion reactions per year and a worldwide market of several billion dollars/year. Nevertheless, PCR still relies on the laborious, time-consuming, and multi-step gel electrophoresis-based detection, which includes gel casting, electrophoretic run, gel staining, and gel visualization. In this work, we propose a "PCR developer", namely a universal one-step, one-tube method, based on controlled aggregation of gold nanoparticles (AuNPs), to detect PCR products by naked eye in few minutes, with no need for any instrumentation. We demonstrated the specificity and sensitivity of the PCR developer on different model targets, suitable for a qualitative detection in real-world diagnostics (i.e., gene rearrangements, genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

  8. Particle creation by naked singularities in higher dimensions

    SciTech Connect

    Miyamoto, Umpei; Nemoto, Hiroya; Shimano, Masahiro

    2011-04-15

    Recently, the possibility was pointed out by one of the present authors and his collaborators that an effective naked singularity referred to as ''a visible border of spacetime'' is generated by high-energy particle collision in the context of large extra dimensions or TeV-scale gravity. In this paper, we investigate the particle creation by a naked singularity in general dimensions, while adopting a model in which a marginally naked singularity forms in the collapse of a homothetic lightlike pressureless fluid. We find that the spectrum deviates from that of Hawking radiation due to scattering near the singularity but can be recast in quasithermal form. The temperature is always higher than that of Hawking radiation of a same-mass black hole, and can be arbitrarily high depending on a parameter in the model. This implies that, in principle, the naked singularity may be distinguished from a black hole in collider experiments.

  9. Quantum naked singularities in 2D dilaton gravity

    NASA Astrophysics Data System (ADS)

    Vaz, Cenalo; Witten, Louis

    1997-02-01

    Roughly speaking, naked singularities are singularities that may be seen by timelike observers. The Cosmic Censorship conjecture forbids their existence by stating that a reasonable system of energy will not, under reasonable conditions, collapse into a naked singularity. There are however many (classical) counter-examples to this conjecture in the literature. We propose a defense of the conjecture through the quantum theory. We will show that the Hawking effect and the accompanying back reaction, when consistently applied to naked singularities in two-dimensional models of dilaton gravity with matter and a cosmological constant, prevent their formation by causing them to explode or to emit radiation catastrophically. This contrasts with black holes which radiate slowly. If this phenomenon is reproduced in the four-dimensional world, the radiation accompanying the formation of naked singularities should have observable consequences.

  10. Postpolio syndrome: poliovirus persistence is involved in the pathogenesis.

    PubMed

    Julien, J; Leparc-Goffart, I; Lina, B; Fuchs, F; Foray, S; Janatova, I; Aymard, M; Kopecka, H

    1999-06-01

    The pathogenesis of the postpolio syndrome (PPS) remains unclear. In this study we looked for poliovirus (PV) persistence in the CSF of 20 patients with PPS, in a control group including 20 patients with unrelated neurological diseases, and in 7 patients with stable poliomyelitis sequelae. CSF samples and sera were examined using reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of PV or other enterovirus genomes; this assay allows the detection from as little as 1 fg viral RNA. Sequencing of amplified products from 5 patients was performed. PV genomic sequences were detected in the CSF of 11 of 20 patients with PPS and in none of the control group. Sequencing in the 5' untranslated region confirmed the presence of mutated PV sequences. These findings suggest that PPS is related to the persistence of PV in the central nervous system. PMID:10431774

  11. Cytotoxic and immunogenic mechanisms of recombinant oncolytic poliovirus.

    PubMed

    Brown, Michael C; Gromeier, Matthias

    2015-08-01

    An oncolytic virus (OV) based on poliovirus (PV), the highly attenuated polio/rhinovirus recombinant PVSRIPO, may deliver targeted inflammatory cancer cell killing; a principle that is showing promise in clinical trials for recurrent glioblastoma (GBM). The two decisive factors in PVSRIPO anti-tumor efficacy are selective cytotoxicity and its in situ immunogenic imprint. While our work is focused on what constitutes PVSRIPO cancer cytotoxicity, we are also studying how this engenders host immune responses that are vital to tumor regression. We hypothesize that PVSRIPO cytotoxicity and immunogenicity are inextricably linked in essential, complimentary roles that define the anti-neoplastic response. Herein we delineate mechanisms we unraveled to decipher the basis for PVSRIPO cytotoxicity and its immunotherapeutic potential. PMID:26083317

  12. Survival of poliovirus within organic solids during chlorination.

    PubMed Central

    Hejkal, T W; Wellings, F M; LaRock, P A; Lewis, A L

    1979-01-01

    Poliovirus in fecal homogenates was used to determine the protection against inactivation by chlorination afforded virus that was occluded within particulates. Virus that was closely associated with or occluded within small fecal particulates was protected. A fourfold increase in combined residual chlorine was required to achieve the same degree of inactivation for occluded virus as for free or secondarily adsorbed virus. A combined chlorine residual of 6.6 mg/liter was necessary to achieve 50% inactivation in 15 min at pH 8.0 and 22 degrees C in a particulate suspension containing occluded virus compared to 1.4 mg/liter for free virus. These differences were found to be relatively small compared to differences due to the presence of dissolved organics or between free and combined chlorine residuals. The results suggest different mechanisms of protection due to adsorption and occlusion. PMID:225993

  13. The Sabin live poliovirus vaccination trials in the USSR, 1959.

    PubMed Central

    Horstmann, D. M.

    1991-01-01

    Widespread use of the Sabin live attenuated poliovirus vaccine has had tremendous impact on the disease worldwide, virtually eliminating it from a number of countries, including the United States. Early proof of its safety and effectiveness was presented in 1959 by Russian investigators, who had staged massive trials in the USSR, involving millions of children. Their positive results were at first viewed in the United States and elsewhere with some skepticism, but the World Health Organization favored proceeding with large-scale trials, and responded to the claims made by Russian scientists by sending a representative to the USSR to review in detail the design and execution of the vaccine programs and the reliability of their results. The report that followed was a positive endorsement of the findings and contributed to the acceptance of the Sabin vaccine in the United States, where it has been the polio vaccine of choice since the mid-1960s. PMID:1814062

  14. A survey for neutralizing antibodies to the three types of poliovirus among children in Maiduguri, Nigeria.

    PubMed

    Baba, M M; Haruna, B A; Ogunmola, O; Ambe, J P; Shidali, N N; Oderinde, B; Marcello, A; Talle, M

    2012-04-01

    The milestone in polio eradication program is to protect effectively children aged 0-5 years against the three serotypes of poliovirus. It became necessary to measure the level of neutralizing antibodies to the three poliovirus types in an endemic State in Nigeria. Neutralizing antibodies to the poliovirus types among children aged 0-5 years was estimated using micro neutralization assay. Of 129 children, 99 (76.8%), 95 (73.6%), and 95 (73.6%) had neutralizing antibodies with the geometric mean titer of 42.7, 31.3, and 33.2 for the poliovirus type 1, 2, and 3, respectively. Fifty-three percent of the children were protected against the three types of poliovirus. Combination of poliovirus types 1 and 2, 1 and 3, and 2 and 3 were neutralized by 62.8, 58.9, and 61.2% of the children studied, respectively. Only poliovirus type 1 induced antibody titres ≥1:1,024. The number of children with neutralizing antibodies after receiving three doses was significantly higher than those who received one or two doses of oral polio vaccine (P ≤ 0.05). However, those who received more than three doses of oral polio vaccine showed no significant difference in their antibody response. The existence of immunity gap poses a risk of re-emergence of the paralytic poliovirus. The existence of unimmunized and unprotected children along with high birth rate could impede the success of polio vaccination in Nigeria. Elimination of non-compliance to polio vaccine, promotion of health education and documented evidence of vaccination of each child with the parents may facilitate the success of polio eradication program in Nigeria. PMID:22337311

  15. Naked-eye observations of Venus in daylight

    NASA Astrophysics Data System (ADS)

    Ellis, E. L.

    1995-12-01

    It is not easy to find Venus with the naked eye in broad daylight. Trying to locate a small white point of light in a light blue background without optical aid is extremely difficult. However under ideal conditions, when the sky is a deep blue and Venus is at or near its maximum brilliancy, it should not be too difficult for a skilled observer with good eyesight to spot Venus at midday, with the naked eye.

  16. Functional analysis of the plasmid pM4 replicon from Lactobacillus plantarum M4: determination of the minimal replicon and functionality identification of the putative sso.

    PubMed

    Yin, Sheng; Hao, Yanling; Zhai, Zhengyuan; Zhang, Wei; Zhou, Hui; Wang, Guohong; Shi, Xianli; Luo, Yunbo

    2009-11-01

    In order to determine the minimal replicon and the single strand origin (sso) of the plasmid pM4, different fragments of pM4 were amplified by polymerase chain reaction (PCR) and cloned into pBEm, a replication probe vector for Lactobacillus. The deletion analysis results showed that the minimal replicon of pM4 could be determined within a 1280bp fragment consisting of double strand origin (dso) and rep gene encoding replication protein. Based on plasmid segregation stability assay and its ability to convert single-stranded DNA (ssDNA) to double-stranded DNA (dsDNA) by Southern hybridization, an sso of replication was located at nucleotides -118-92 in the plasmid pM4, about 300bp upstream of dso. In addition, the host range assay indicated that plasmid pM4 could replicate in L. casei 05-21, L. rhamnosus AS 1.2466(T) and L. plantarum 05-19 of all the tested Lactobacillus strains. Analysis of the pM4 replicon will allow its use in constructing a food-grade vector for application in food industry. PMID:19651154

  17. Complex Dynamic Development of Poliovirus Membranous Replication Complexes

    PubMed Central

    Nair, Vinod; Hansen, Bryan T.; Hoyt, Forrest H.; Fischer, Elizabeth R.; Ehrenfeld, Ellie

    2012-01-01

    Replication of all positive-strand RNA viruses is intimately associated with membranes. Here we utilize electron tomography and other methods to investigate the remodeling of membranes in poliovirus-infected cells. We found that the viral replication structures previously described as “vesicles” are in fact convoluted, branching chambers with complex and dynamic morphology. They are likely to originate from cis-Golgi membranes and are represented during the early stages of infection by single-walled connecting and branching tubular compartments. These early viral organelles gradually transform into double-membrane structures by extension of membranous walls and/or collapsing of the luminal cavity of the single-membrane structures. As the double-membrane regions develop, they enclose cytoplasmic material. At this stage, a continuous membranous structure may have double- and single-walled membrane morphology at adjacent cross-sections. In the late stages of the replication cycle, the structures are represented mostly by double-membrane vesicles. Viral replication proteins, double-stranded RNA species, and actively replicating RNA are associated with both double- and single-membrane structures. However, the exponential phase of viral RNA synthesis occurs when single-membrane formations are predominant in the cell. It has been shown previously that replication complexes of some other positive-strand RNA viruses form on membrane invaginations, which result from negative membrane curvature. Our data show that the remodeling of cellular membranes in poliovirus-infected cells produces structures with positive curvature of membranes. Thus, it is likely that there is a fundamental divergence in the requirements for the supporting cellular membrane-shaping machinery among different groups of positive-strand RNA viruses. PMID:22072780

  18. Framework for evaluating the risks of paralytic poliomyelitis after global interruption of wild poliovirus transmission.

    PubMed Central

    Aylward, R. Bruce; Cochi, Stephen L.

    2004-01-01

    With the interruption of wild poliovirus transmission globally, the need for new policies to deal with the post-certification era will rapidly arise. New policies will be required in four areas: detection and notification of circulating polioviruses; biocontainment of wild, vaccine-derived and attenuated strains of poliovirus; vaccine stockpiles and response mechanisms; and routine immunization against polioviruses. A common understanding of the potential risks of paralytic poliomyelitis in the post-certification period is essential to the development of these policies. Since 2000, there has been increasing international consensus that the risks of paralytic poliomyelitis in the post-certification era fall into two categories: those due to the continued use of the oral poliovirus vaccine (OPV) and those due to future improper handling of wild polioviruses. The specific risks within both categories have now been defined, and an understanding of the frequency and potential burden of disease associated with each is rapidly improving. This knowledge and clarity have provided a framework that is already proving valuable for identifying research priorities and discussing potential policy options with national authorities. However, this framework must be regarded as a dynamic tool, requiring regular updating as additional information on these risks becomes available through further scientific research, programmatic work, and policy decisions. PMID:15106299

  19. Contribution of Environmental Surveillance Toward Interruption of Poliovirus Transmission in Nigeria, 2012–2015

    PubMed Central

    Johnson Muluh, Ticha; Hamisu, Abdullahi Walla; Craig, Kehinde; Mkanda, Pascal; Andrew, Etsano; Adeniji, Johnson; Akande, Adefunke; Musa, Audu; Ayodeji, Isiaka; Nicksy, Gumede; Banda, Richard; Tegegne, Sisay G.; Nsubuga, Peter; Oyetunji, Ajiboye; Diop, Ousmane; Vaz, Rui G.; Muhammad, Ado J. G.

    2016-01-01

    Background. Cases of paralysis caused by poliovirus have decreased by >99% since the 1988 World Health Assembly's resolution to eradicate polio. The World Health Organization identified environmental surveillance (ES) of poliovirus in the poliomyelitis eradication strategic plan as an activity that can complement acute flaccid paralysis (AFP) surveillance. This article summarizes key public health interventions that followed the isolation of polioviruses from ES between 2012 and 2015. Methods. The grap method was used to collect 1.75 L of raw flowing sewage every 2–4 weeks. Once collected, samples were shipped at 4°C to a polio laboratory for concentration. ES data were then used to guide program implementation. Results. From 2012 to 2015, ES reported 97 circulating vaccine-derived polioviruses (cVDPV2) and 14 wild polioviruses. In 2014 alone, 54 cVDPV type 2 cases and 1 WPV type 1 case were reported. In Sokoto State, 58 cases of AFP were found from a search of 9426 households. A total of 2 252 059 inactivated polio vaccine and 2 460 124 oral polio vaccine doses were administered to children aged <5 year in Borno and Yobe states. Conclusions. This article is among the first from Africa that relates ES findings to key public health interventions (mass immunization campaigns, inactivated polio vaccine introduction, and strengthening of AFP surveillance) that have contributed to the interruption of poliovirus transmission in Nigeria. PMID:26908747

  20. Current polio global eradication and control policy options: perspectives from modeling and prerequisites for oral poliovirus vaccine cessation.

    PubMed

    Thompson, Kimberly M; Tebbens, Radboud J Duintjer

    2012-04-01

    As the Global Polio Eradication Initiative progresses toward the eradication of wild polioviruses, national and global health leaders must still actively consider options for managing poliovirus risks, including risks associated with using oral poliovirus vaccine. Oral poliovirus vaccine continues to represent a highly effective tool, but its use causes noticeable, rare cases of vaccine-associated paralytic polio and with low coverage it can evolve to become circulating vaccine-derived polioviruse that causes outbreaks. National leaders face a wide range of options, but their choices depend in part on global policies. This article explores the current set of global options for poliovirus eradication or control, discusses constraints and prerequisites for their implementation and offers some insights based on dynamic modeling to inform discussions and frame future economic analyses. PMID:22551030

  1. Replicon typing of plasmids encoding resistance to newer beta-lactams.

    PubMed

    Carattoli, Alessandra; Miriagou, Vivi; Bertini, Alessia; Loli, Alexandra; Colinon, Celine; Villa, Laura; Whichard, Jean M; Rossolini, Gian Maria

    2006-07-01

    Polymerase chain reaction-based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems. PMID:16836838

  2. Replicon Typing of Plasmids Encoding Resistance to Newer β-Lactams

    PubMed Central

    Miriagou, Vivi; Bertini, Alessia; Loli, Alexandra; Colinon, Celine; Villa, Laura; Whichard, Jean M.; Rossolini, Gian Maria

    2006-01-01

    Polymerase chain reaction–based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems. PMID:16836838

  3. [IncP-7 plasmids' classification based on structural diversity of their basic replicons].

    PubMed

    Volkova, O V; Panov, A V; Kosheleva, I A; Boronin, A M

    2013-01-01

    The structural diversity of basic replicons and repB gene of the IncP-7 plasmids' collection was firstly assessed on the basis of PCR, restriction analysis and partial sequencing. It has been revealed that DNA fragment containing gene for UvrD-like helicase RepB is a part of all known P-7 replicons, but often serves as hot place for diverse IS-elements invasion. The first system of P-7 plasmids' classification has been worked out on the basis of determined repA-oriV-par WABC nucleotide divergency. Most degradation plasmids established to be belonging to large beta-subgroup, streptomycin resistance plasmid Rms148 (IncP-7 archetype)--to alpha-subgroup, carbazole degradation plasmid pCAR1 and NAH/SAL-plasmids from pY-line (Yamal oil deposits)--to gamma-subgroup and CAP-plasmid pBS270 with potentially reduced P-7 replicon--to delta-subgroup. It has been observed that the type of IncP-7 basic replicon molecular organization does not correlate with fixed phenotypic character in most cases, that is plasmids encoding different phenotypic markers could be members of the same P-7 subgroup. PMID:23808156

  4. Replicon typing of virulence plasmids of enterotoxigenic Escherichia coli isolates from cattle.

    PubMed Central

    Mainil, J G; Bex, F; Dreze, P; Kaeckenbeeck, A; Couturier, M

    1992-01-01

    Plasmid DNA hybridization with probes for virulence factors used for basic replicons of plasmids was used to identify the virulence plasmids of a collection of enterotoxigenic Escherichia coli isolates from cattle. The virulence probes were derived from the genes coding for the heat-stable enterotoxin STaP and for the F5 (K99) and F41 fimbrial adhesins. The replicon probes were derived from 16 different basic replicons of plasmids (probes repFIA, repFIB, repFIC, repFIIA, repI1, repHI1, repHI2, repL/M, repN, repP, repQ, repT, repU, repW, repX, and repY). The virulence genes coding for the STaP enterotoxin and for the F5 adhesin were located on a single plasmid band in each isolate. The sizes of most of these virulence plasmids were from 65 to 95 MDa. The F41 probe failed to hybridize with any plasmid band. The virulence plasmids had multireplicon types typical of plasmids of the IncF groups. The most common basic replicon association was the triple RepFIA-RepFIB-RepFIC family association. Images PMID:1639505

  5. Cell-Free Replication of the Hepatitis C Virus Subgenomic Replicon

    PubMed Central

    Ali, Naushad; Tardif, Keith D.; Siddiqui, Aleem

    2002-01-01

    The hepatitis C virus (HCV) contains a plus-strand RNA genome. The 5′ noncoding region (NCR) of the viral genome functions as an internal ribosome entry site, and its unique 3′ NCR is required for the assembly of the replication complex during initiation of HCV RNA replication. Lohmann et al. (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilman, and R. Batenschlager, Science 285:110-113, 1999) developed a subgenomic HCV replicon system, which represents an important tool in studying HCV replication in cultured cells. In this study, we describe a cell-free replication system that utilizes cytoplasmic lysates prepared from Huh-7 cells harboring the HCV subgenomic replicons. These lysates, which contain ribonucleoprotein complexes associated with cellular membranes, were capable of incorporating [α32P]CTP into newly synthesized RNA from subgenomic replicons in vitro. Replicative forms (RFs) and replicative intermediates (RIs) were synthesized from the endogenous HCV RNA templates. Consistent with previous observations, RFs were found to be resistant to RNase A digestion, whereas RIs were sensitive to RNase treatment. The radiolabeled HCV RF-RI complexes contained both minus and plus strands and were specific to the lysates derived from replicon-expressing cells. The availability of a cell-free replication system offers opportunities to probe the mechanism(s) of HCV replication. It also provides a novel assay for potential therapeutic agents. PMID:12414942

  6. Do sister forks of bidirectionally growing replicons proceed at unequal rates

    SciTech Connect

    Dubey, D.D.; Raman, R.

    1987-02-01

    DNA fibre autoradiography in different tissues of the rodents Bandicota bengalensis and Nesokia indica reveals a high frequency of such bidirectionally replicating replicons whose sister hot tracks are of unequal size. These results suggest intrarepliconic difference in the rates of fork migration in the two directions.

  7. Hepatitis C Virus Subgenomic Replicons Induce Endoplasmic Reticulum Stress Activating an Intracellular Signaling Pathway

    PubMed Central

    Tardif, Keith D.; Mori, Kazutoshi; Siddiqui, Aleem

    2002-01-01

    Hepatitis C virus (HCV) replicates from a ribonucleoprotein (RNP) complex that is associated with the endoplasmic reticulum (ER) membrane. The replication activities of the HCV subgenomic replicon are shown here to induce ER stress. In response to this stress, cells expressing HCV replicons induce the unfolded protein response (UPR), an ER-to-nucleus intracellular signaling pathway. The UPR is initiated by the proteolytic cleavage of a transmembrane protein, ATF6. The resulting cytoplasmic protein fragment of ATF6 functions as a transcription factor in the nucleus and activates selective genes required for an ER stress response. ATF6 activation leads to increased transcriptional levels of GRP78, an ER luminal chaperone protein. However, the overall level of GRP78 protein is decreased. While ER stress is also known to affect translational attenuation, cells expressing HCV replicons have lower levels of phosphorylation of the α subunit of eukaryotic initiation factor 2. Interestingly, cap-independent internal ribosome entry site-mediated translation directed by the 5′ noncoding region of HCV and GRP78 is activated in cells expressing HCV replicons. These studies provide insight into the effects of HCV replication on intracellular events and the mechanisms underlying liver pathogenesis. PMID:12097557

  8. Pea (Pisum sativum) cells arrested in G2 have nascent DNA with breaks between replicons and replication clusters

    SciTech Connect

    Van't Hof, J.

    1980-01-01

    DNA fiber autoradiography and alkaline sucrose sedimentation of DNA of cultured pea-root cells (Pisum sativum) arrested in G2 by carbohydrate starvation demonstrated that nascent DNA molecules of replicon (16 to 27 x 10/sup 6/D) and apparent cluster (approx. 330 x 10/sup 6/D) size were not joined. That the arrested cells were in G2 was confirmed by single-cell autoradiography and cytophotometry. In pea there are about 18 replicons per average cluster, 4.2 x 10/sup 3/ clusters, and 7.7 x 10/sup 4/ replicons per genome.

  9. Synthetic virus seeds for improved vaccine safety: Genetic reconstruction of poliovirus seeds for a PER.C6 cell based inactivated poliovirus vaccine.

    PubMed

    Sanders, Barbara P; Edo-Matas, Diana; Papic, Natasa; Schuitemaker, Hanneke; Custers, Jerome H H V

    2015-10-13

    Safety of vaccines can be compromised by contamination with adventitious agents. One potential source of adventitious agents is a vaccine seed, typically derived from historic clinical isolates with poorly defined origins. Here we generated synthetic poliovirus seeds derived from chemically synthesized DNA plasmids encoding the sequence of wild-type poliovirus strains used in marketed inactivated poliovirus vaccines. The synthetic strains were phenotypically identical to wild-type polioviruses as shown by equivalent infectious titers in culture supernatant and antigenic content, even when infection cultures are scaled up to 10-25L bioreactors. Moreover, the synthetic seeds were genetically stable upon extended passaging on the PER.C6 cell culture platform. Use of synthetic seeds produced on the serum-free PER.C6 cell platform ensures a perfectly documented seed history and maximum control over starting materials. It provides an opportunity to maximize vaccine safety which increases the prospect of a vaccine end product that is free from adventitious agents. PMID:26362098

  10. Preventing Vaccine-Derived Poliovirus Emergence during the Polio Endgame.

    PubMed

    Pons-Salort, Margarita; Burns, Cara C; Lyons, Hil; Blake, Isobel M; Jafari, Hamid; Oberste, M Steven; Kew, Olen M; Grassly, Nicholas C

    2016-07-01

    Reversion and spread of vaccine-derived poliovirus (VDPV) to cause outbreaks of poliomyelitis is a rare outcome resulting from immunisation with the live-attenuated oral poliovirus vaccines (OPVs). Global withdrawal of all three OPV serotypes is therefore a key objective of the polio endgame strategic plan, starting with serotype 2 (OPV2) in April 2016. Supplementary immunisation activities (SIAs) with trivalent OPV (tOPV) in advance of this date could mitigate the risks of OPV2 withdrawal by increasing serotype-2 immunity, but may also create new serotype-2 VDPV (VDPV2). Here, we examine the risk factors for VDPV2 emergence and implications for the strategy of tOPV SIAs prior to OPV2 withdrawal. We first developed mathematical models of VDPV2 emergence and spread. We found that in settings with low routine immunisation coverage, the implementation of a single SIA increases the risk of VDPV2 emergence. If routine coverage is 20%, at least 3 SIAs are needed to bring that risk close to zero, and if SIA coverage is low or there are persistently "missed" groups, the risk remains high despite the implementation of multiple SIAs. We then analysed data from Nigeria on the 29 VDPV2 emergences that occurred during 2004-2014. Districts reporting the first case of poliomyelitis associated with a VDPV2 emergence were compared to districts with no VDPV2 emergence in the same 6-month period using conditional logistic regression. In agreement with the model results, the odds of VDPV2 emergence decreased with higher routine immunisation coverage (odds ratio 0.67 for a 10% absolute increase in coverage [95% confidence interval 0.55-0.82]). We also found that the probability of a VDPV2 emergence resulting in poliomyelitis in >1 child was significantly higher in districts with low serotype-2 population immunity. Our results support a strategy of focused tOPV SIAs before OPV2 withdrawal in areas at risk of VDPV2 emergence and in sufficient number to raise population immunity above the

  11. Preventing Vaccine-Derived Poliovirus Emergence during the Polio Endgame

    PubMed Central

    Burns, Cara C.; Lyons, Hil; Blake, Isobel M.; Oberste, M. Steven; Kew, Olen M.; Grassly, Nicholas C.

    2016-01-01

    Reversion and spread of vaccine-derived poliovirus (VDPV) to cause outbreaks of poliomyelitis is a rare outcome resulting from immunisation with the live-attenuated oral poliovirus vaccines (OPVs). Global withdrawal of all three OPV serotypes is therefore a key objective of the polio endgame strategic plan, starting with serotype 2 (OPV2) in April 2016. Supplementary immunisation activities (SIAs) with trivalent OPV (tOPV) in advance of this date could mitigate the risks of OPV2 withdrawal by increasing serotype-2 immunity, but may also create new serotype-2 VDPV (VDPV2). Here, we examine the risk factors for VDPV2 emergence and implications for the strategy of tOPV SIAs prior to OPV2 withdrawal. We first developed mathematical models of VDPV2 emergence and spread. We found that in settings with low routine immunisation coverage, the implementation of a single SIA increases the risk of VDPV2 emergence. If routine coverage is 20%, at least 3 SIAs are needed to bring that risk close to zero, and if SIA coverage is low or there are persistently “missed” groups, the risk remains high despite the implementation of multiple SIAs. We then analysed data from Nigeria on the 29 VDPV2 emergences that occurred during 2004−2014. Districts reporting the first case of poliomyelitis associated with a VDPV2 emergence were compared to districts with no VDPV2 emergence in the same 6-month period using conditional logistic regression. In agreement with the model results, the odds of VDPV2 emergence decreased with higher routine immunisation coverage (odds ratio 0.67 for a 10% absolute increase in coverage [95% confidence interval 0.55−0.82]). We also found that the probability of a VDPV2 emergence resulting in poliomyelitis in >1 child was significantly higher in districts with low serotype-2 population immunity. Our results support a strategy of focused tOPV SIAs before OPV2 withdrawal in areas at risk of VDPV2 emergence and in sufficient number to raise population immunity

  12. Orderly Replication and Segregation of the Four Replicons of Burkholderia cenocepacia J2315

    PubMed Central

    Kamgoué, Alain; Murray, Heath; Pasta, Franck

    2016-01-01

    Bacterial genomes typically consist of a single chromosome and, optionally, one or more plasmids. But whole-genome sequencing reveals about ten per-cent of them to be multipartite, with additional replicons which by size and indispensability are considered secondary chromosomes. This raises the questions of how their replication and partition is managed without compromising genome stability and of how such genomes arose. Vibrio cholerae, with a 1 Mb replicon in addition to its 3 Mb chromosome, is the only species for which maintenance of a multipartite genome has been investigated. In this study we have explored the more complex genome of Burkholderia cenocepacia (strain J2315). It comprises an extra replicon (c2) of 3.21 Mb, comparable in size to the3.87Mb main chromosome (c1), another extra replicon(c3) of 0.87 Mb and a plasmid of 0.09 Mb. The replication origin of c1 is typically chromosomal and those of c2 and c3 are plasmid-like; all are replicated bidirectionally. Fluorescence microscopy of tagged origins indicates that all initiate replication at mid-cell and segregate towards the cell quarter positions sequentially, c1-c2-p1/c3. c2 segregation is as well-phased with the cell cycle as c1, implying that this plasmid-like origin has become subject to regulation not typical of plasmids; in contrast, c3 segregates more randomly through the cycle. Disruption of individual Par systems by deletion of parAB or by addition of parS sites showed each Par system to govern the positioning of its own replicon only. Inactivation of c1, c2 and c3 Par systems not only reduced growth rate, generated anucleate cells and compromised viability but influenced processes beyond replicon partition, notably regulation of replication, chromosome condensation and cell size determination. In particular, the absence of the c1 ParA protein altered replication of all three chromosomes, suggesting that the partition system of the main chromosome is a major participant in the choreography of

  13. Orderly Replication and Segregation of the Four Replicons of Burkholderia cenocepacia J2315.

    PubMed

    Du, Wen-Li; Dubarry, Nelly; Passot, Fanny M; Kamgoué, Alain; Murray, Heath; Lane, David; Pasta, Franck

    2016-07-01

    Bacterial genomes typically consist of a single chromosome and, optionally, one or more plasmids. But whole-genome sequencing reveals about ten per-cent of them to be multipartite, with additional replicons which by size and indispensability are considered secondary chromosomes. This raises the questions of how their replication and partition is managed without compromising genome stability and of how such genomes arose. Vibrio cholerae, with a 1 Mb replicon in addition to its 3 Mb chromosome, is the only species for which maintenance of a multipartite genome has been investigated. In this study we have explored the more complex genome of Burkholderia cenocepacia (strain J2315). It comprises an extra replicon (c2) of 3.21 Mb, comparable in size to the3.87Mb main chromosome (c1), another extra replicon(c3) of 0.87 Mb and a plasmid of 0.09 Mb. The replication origin of c1 is typically chromosomal and those of c2 and c3 are plasmid-like; all are replicated bidirectionally. Fluorescence microscopy of tagged origins indicates that all initiate replication at mid-cell and segregate towards the cell quarter positions sequentially, c1-c2-p1/c3. c2 segregation is as well-phased with the cell cycle as c1, implying that this plasmid-like origin has become subject to regulation not typical of plasmids; in contrast, c3 segregates more randomly through the cycle. Disruption of individual Par systems by deletion of parAB or by addition of parS sites showed each Par system to govern the positioning of its own replicon only. Inactivation of c1, c2 and c3 Par systems not only reduced growth rate, generated anucleate cells and compromised viability but influenced processes beyond replicon partition, notably regulation of replication, chromosome condensation and cell size determination. In particular, the absence of the c1 ParA protein altered replication of all three chromosomes, suggesting that the partition system of the main chromosome is a major participant in the choreography of

  14. EFFECT OF ENDOTOXIN ON CELLS AND ON THEIR RESPONSE TO INFECTION BY POLIOVIRUSES1

    PubMed Central

    Murphy, William H.; Wisner, Carolyn

    1962-01-01

    Murphy, W. H. (The University of Michigan, Ann Arbor) and C. Wisner. Effect of endotoxin on cells and on their response to infection by polioviruses. J. Bacteriol. 83:649–662. 1962.—The effect of lipopolysaccharide on HeLa-S3, HeLa-Gey, Chang-liver, Maben, and L strain mouse fibroblasts was studied. The liminal dose of endotoxin for the human epithelial cell strains was approximately 250 μg/ml, and their order of sensitivity to endotoxin was: Chang-liver, HeLa-Gey, HeLa-S3, and Maben, the latter being the most resistant. Endotoxin at concentrations exceeding 100 μg/ml was cytotoxic to the L strain of mouse fibroblasts and caused them to markedly agglutinate. Cytotoxic response of cells to endotoxin was not characterized by cell lysis, but by distinctive nuclear changes. In an attempt to demonstrate the metabolic induction of the latent infection of cell cultures by a noncytopathic variant of poliovirus, endotoxin was added at maximal subliminal concentration to cell cultures totally, partially, or fully susceptible to virus. Endotoxin caused a slight but consistent accelerative cytopathic response of cells to infection by cytopathic poliovirus, but failed to induce cytopathic response to infection by submoderate (noncytopathic) poliovirus. Although endotoxin slightly suppressed yields of poliovirus from cells, it did not affect the plating efficiency of virus on cell monolayers. Images PMID:14477444

  15. Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies.

    PubMed Central

    Emini, E A; Jameson, B A; Lewis, A J; Larsen, G R; Wimmer, E

    1982-01-01

    Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites. PMID:6183443

  16. Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR.

    PubMed

    Burns, Cara C; Kilpatrick, David R; Iber, Jane C; Chen, Qi; Kew, Olen M

    2016-01-01

    Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures. PMID:26983735

  17. Characterization and replication mode determination of the minimal replicon of Tetragenococcus halophila ATCC33315 plasmid pUCL287.

    PubMed

    Benachour, A; Frère, J; Boutibonnes, P; Auffray, Y

    1995-01-01

    pUCL287 is a cryptic plasmid of Tetragenococcus halophila (formerly Pediococcus halophilus) ATCC33315 of relatively small size (8.7 kb). Its minimal replicon was located on a 1235 bp MamI-EcoRI fragment. This minimal replicon contains a non-translated region, followed by a gene encoding a putative 311 amino acid protein. Deletion experiments showed that the non-translated region corresponds to the replication origin. Determination of the replication mode was carried out in Enterococcus faecalis JH2-2 harboring pUCL287 minimal replicon. The replicating intermediates detected revealed that pUCL287 minimal replicon follows a bidirectional theta replicating mode. PMID:8824766

  18. Dengue Virus Reporter Replicon is a Valuable Tool for Antiviral Drug Discovery and Analysis of Virus Replication Mechanisms.

    PubMed

    Kato, Fumihiro; Hishiki, Takayuki

    2016-01-01

    Dengue, the most prevalent arthropod-borne viral disease, is caused by the dengue virus (DENV), a member of the Flaviviridae family, and is a considerable public health threat in over 100 countries, with 2.5 billion people living in high-risk areas. However, no specific antiviral drug or licensed vaccine currently targets DENV infection. The replicon system has all the factors needed for viral replication in cells. Since the development of replicon systems, transient and stable reporter replicons, as well as reporter viruses, have been used in the study of various virological aspects of DENV and in the identification of DENV inhibitors. In this review, we summarize the DENV reporter replicon system and its applications in high-throughput screening (HTS) for identification of anti-DENV inhibitors. We also describe the use of this system in elucidation of the mechanisms of virus replication and viral dynamics in vivo and in vitro. PMID:27164125

  19. Dengue Virus Reporter Replicon is a Valuable Tool for Antiviral Drug Discovery and Analysis of Virus Replication Mechanisms

    PubMed Central

    Kato, Fumihiro; Hishiki, Takayuki

    2016-01-01

    Dengue, the most prevalent arthropod-borne viral disease, is caused by the dengue virus (DENV), a member of the Flaviviridae family, and is a considerable public health threat in over 100 countries, with 2.5 billion people living in high-risk areas. However, no specific antiviral drug or licensed vaccine currently targets DENV infection. The replicon system has all the factors needed for viral replication in cells. Since the development of replicon systems, transient and stable reporter replicons, as well as reporter viruses, have been used in the study of various virological aspects of DENV and in the identification of DENV inhibitors. In this review, we summarize the DENV reporter replicon system and its applications in high-throughput screening (HTS) for identification of anti-DENV inhibitors. We also describe the use of this system in elucidation of the mechanisms of virus replication and viral dynamics in vivo and in vitro. PMID:27164125

  20. Progress in the development of poliovirus antiviral agents and their essential role in reducing risks that threaten eradication.

    PubMed

    McKinlay, Mark A; Collett, Marc S; Hincks, Jeffrey R; Oberste, M Steven; Pallansch, Mark A; Okayasu, Hiromasa; Sutter, Roland W; Modlin, John F; Dowdle, Walter R

    2014-11-01

    Chronic prolonged excretion of vaccine-derived polioviruses by immunodeficient persons (iVDPV) presents a personal risk of poliomyelitis to the patient as well as a programmatic risk of delayed global eradication. Poliovirus antiviral drugs offer the only mitigation of these risks. Antiviral agents may also have a potential role in the management of accidental exposures and in certain outbreak scenarios. Efforts to discover and develop poliovirus antiviral agents have been ongoing in earnest since the formation in 2007 of the Poliovirus Antivirals Initiative. The most advanced antiviral, pocapavir (V-073), is a capsid inhibitor that has recently demonstrated activity in an oral poliovirus vaccine human challenge model. Additional antiviral candidates with differing mechanisms of action continue to be profiled and evaluated preclinically with the goal of having 2 antivirals available for use in combination to treat iVDPV excreters. PMID:25316866

  1. New Generation of Inactivated Poliovirus Vaccines for Universal Immunization After Eradication of Poliomyelitis

    PubMed Central

    Chumakov, Konstantin; Ehrenfeld, Ellie

    2008-01-01

    Twenty years of global polio eradication efforts may soon eliminate wild-type poliovirus transmission. However, new information about poliovirus learned during this campaign, as well as the political realities of a modern world demand that universal immunity against poliomyelitis be maintained even after wild poliovirus is eradicated. Although two excellent vaccines have proven highly effective in the past, neither the live nor current inactivated products are optimal for use in the post-eradication setting. Therefore, concerted efforts are urgently needed to develop a new generation of vaccine that is risk-free and affordable and can be produced on a global scale. Here we discuss the desired properties and ways to create a new polio vaccine. PMID:18990066

  2. Development of a poliovirus neutralization test with poliovirus pseudovirus for measurement of neutralizing antibody titer in human serum.

    PubMed

    Arita, Minetaro; Iwai, Masae; Wakita, Takaji; Shimizu, Hiroyuki

    2011-11-01

    In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying poliovirus (PV) from the stool samples from acute flaccid paralysis (AFP) cases. In recent years, reestablishment of PV circulation in countries where PV was previously eliminated has occurred because of decreased herd immunity, possibly due to poor vaccination coverage. To monitor the vulnerability of countries to PV circulation, surveillance of neutralizing-antibody titers against PV in susceptible populations is essential in the end game of the polio eradication program. In this study, we have developed a PV neutralization test with type 1, 2, and 3 PV pseudoviruses to determine the neutralizing-antibody titer against PV in human serum samples. With this test, the neutralizing-antibody titer against PV could be determined within 2 days by automated interpretation of luciferase signals without using infectious PV strains. We validated the pseudovirus PV neutralization test with 131 human serum samples collected from a wide range of age groups (ages 1 to >60 years) by comparison with a conventional neutralization test. We found good correlation in the neutralizing-antibody titers determined by these tests. These results suggest that a pseudovirus PV neutralization test would serve as a safe and simple procedure for the measurement of the neutralizing-antibody titer against PV. PMID:21880850

  3. Three Replicons of Rhizobium sp. Strain NGR234 Harbor Symbiotic Gene Sequences

    PubMed Central

    Flores, Margarita; Mavingui, Patrick; Girard, Lourdes; Perret, Xavier; Broughton, William J.; Martínez-Romero, Esperanza; Dávila, Guillermo; Palacios, Rafael

    1998-01-01

    Rhizobium sp. strain NGR234 contains three replicons: the symbiotic plasmid or pNGR234a, a megaplasmid (pNGR234b), and the chromosome. Symbiotic gene sequences not present in pNGR234a were analyzed by hybridization. DNA sequences homologous to the genes fixLJKNOPQGHIS were found on the chromosome, while sequences homologous to nodPQ and exoBDFLK were found on pNGR234b. PMID:9811668

  4. Hepatitis C Virus Genotype 5a Subgenomic Replicons for Evaluation of Direct-Acting Antiviral Agents

    PubMed Central

    Wose Kinge, Constance N.; Espiritu, Christine; Prabdial-Sing, Nishi; Sithebe, Nomathamsaqa Patricia

    2014-01-01

    Hepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy. In vitro replication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to replicate in Huh-7.5 cells. However, the inclusion of the S2205I amino acid substitution, a cell culture-adaptive change originally described for a genotype 1b replicon, resulted in a small number of G418-resistant cell colonies. HCV RNA replication in these cells was confirmed by quantification of viral RNA and detection of the nonstructural protein NS5A. Sequence analysis of the viral RNAs isolated from multiple independent cell clones revealed the presence of several nonsynonymous mutations, which were localized mainly in the NS3 protein. These mutations, when introduced back into the parental backbone, significantly increased colony formation. To facilitate convenient monitoring of HCV RNA replication levels, the mutant with the highest replication level was further modified to express a fusion protein of firefly luciferase and neomycin phosphotransferase. Using such replicons from genotypes 1a, 1b, 2a, 3a, 4a, and 5a, we compared the effects of various HCV inhibitors on their replication. In conclusion, we have established an in vitro replication system for HCV genotype 5a, which will be useful for the development of pan-genotype anti-HCV compounds. PMID:24982066

  5. Use of Recombinant Virus Replicon Particles for Vaccination against Mycobacterium ulcerans Disease

    PubMed Central

    Bolz, Miriam; Kerber, Sarah; Zimmer, Gert; Pluschke, Gerd

    2015-01-01

    Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a necrotizing disease of the skin and subcutaneous tissue, which is most prevalent in rural regions of West African countries. The majority of clinical presentations seen in patients are ulcers on limbs that can be treated by eight weeks of antibiotic therapy. Nevertheless, scarring and permanent disabilities occur frequently and Buruli ulcer still causes high morbidity. A vaccine against the disease is so far not available but would be of great benefit if used for prophylaxis as well as therapy. In the present study, vesicular stomatitis virus-based RNA replicon particles encoding the M. ulcerans proteins MUL2232 and MUL3720 were generated and the expression of the recombinant antigens characterized in vitro. Immunisation of mice with the recombinant replicon particles elicited antibodies that reacted with the endogenous antigens of M. ulcerans cells. A prime-boost immunization regimen with MUL2232-recombinant replicon particles and recombinant MUL2232 protein induced a strong immune response but only slightly reduced bacterial multiplication in a mouse model of M. ulcerans infection. We conclude that a monovalent vaccine based on the MUL2232 antigen will probably not sufficiently control M. ulcerans infection in humans. PMID:26275222

  6. chr genes from adaptive replicons are responsible for chromate resistance by Burkholderia xenovorans LB400.

    PubMed

    Reyes-Gallegos, Rosa I; Ramírez-Díaz, Martha I; Cervantes, Carlos

    2016-03-01

    The chromate ion transporter (CHR) superfamily includes proteins that confer chromate resistance by extruding toxic chromate ions from cytoplasm. Burkholderia xenovorans strain LB400 encodes six CHR homologues in its multireplicon genome and has been reported as highly chromate-resistant. The objective of this work was to analyze the involvement of chr redundant genes in chromate resistance by LB400. It was found that B. xenovorans plant rhizosphere strains lacking the megaplasmid are chromate-sensitive, suggesting that the chr gene present in this replicon is responsible for the chromate-resistance phenotype of the LB400 strain. Transformation of a chromate-sensitive B. xenovorans strain with each of the six cloned LB400 chr genes showed that genes from 'adaptive replicons' (chrA1b and chr1NCb from chromosome 2 and chrA2 from the megaplasmid) conferred higher chromate resistance levels than chr genes from 'central' chromosome 1 (chrA1a, chrA6, and chr1NCa). An LB400 insertion mutant affected in the chrA2 gene displayed a chromate-sensitive phenotype, which was fully reverted by transferring the chrA2 wild-type gene, and partially reverted by chrA1b or chr1NCb genes. These data indicate that chr genes from adaptive replicons, mainly chrA2 from the megaplasmid, are responsible for the B. xenovorans LB400 chromate-resistance phenotype. PMID:26873556

  7. Fractional-Dose Inactivated Poliovirus Vaccine Immunization Campaign - Telangana State, India, June 2016.

    PubMed

    Bahl, Sunil; Verma, Harish; Bhatnagar, Pankaj; Haldar, Pradeep; Satapathy, Asish; Kumar, K N Arun; Horton, Jennifer; Estivariz, Concepcion F; Anand, Abhijeet; Sutter, Roland

    2016-01-01

    Wild poliovirus type 2 was declared eradicated in September 2015 (1). In April 2016, India, switched from use of trivalent oral poliovirus vaccine (tOPV; containing types 1, 2, and 3 polio vaccine viruses), to bivalent OPV (bOPV; containing types 1 and 3), as part of a globally synchronized initiative to withdraw Sabin poliovirus type 2 vaccine. Concurrently, inactivated poliovirus vaccine (IPV) was introduced into India's routine immunization program to maintain an immunity base that would mitigate the number of paralytic cases in the event of epidemic transmission of poliovirus type 2 (2,3). After cessation of use of type 2 Sabin vaccine, any reported isolation of vaccine-derived poliovirus type 2 (VDPV2) would be treated as a public health emergency and might need outbreak response with monovalent type 2 oral vaccine, IPV, or both (4). In response to identification of a VDPV2 isolate from a sewage sample collected in the southern state of Telangana in May 2016, India conducted a mass vaccination campaign in June 2016 using an intradermal fractional dose (0.1 ml) of IPV (fIPV). Because of a global IPV supply shortage, fIPV, which uses one fifth of regular intramuscular (IM) dose administered intradermally, has been recommended as a response strategy for VDPV2 (5). Clinical trials have demonstrated that fIPV is highly immunogenic (6,7). During the 6-day campaign, 311,064 children aged 6 weeks-3 years were vaccinated, achieving an estimated coverage of 94%. With appropriate preparation, an emergency fIPV response can be promptly and successfully implemented. Lessons learned from this campaign can be applied to successful implementation of future outbreak responses using fIPV. PMID:27559683

  8. Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine.

    PubMed

    Sanders, Barbara P; de Los Rios Oakes, Isabel; van Hoek, Vladimir; Bockstal, Viki; Kamphuis, Tobias; Uil, Taco G; Song, Yutong; Cooper, Gillian; Crawt, Laura E; Martín, Javier; Zahn, Roland; Lewis, John; Wimmer, Eckard; Custers, Jerome H H V; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

    2016-03-01

    The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4-9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive

  9. Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine

    PubMed Central

    Sanders, Barbara P.; de los Rios Oakes, Isabel; van Hoek, Vladimir; Bockstal, Viki; Kamphuis, Tobias; Uil, Taco G.; Song, Yutong; Cooper, Gillian; Crawt, Laura E.; Martín, Javier; Zahn, Roland; Lewis, John; Wimmer, Eckard; Custers, Jerome H. H. V.; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

    2016-01-01

    The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4–9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive

  10. Naked at Our Age: Talking out Loud about Senior Sex

    ERIC Educational Resources Information Center

    Davis, Melanie

    2012-01-01

    "Naked at Our Age" is an excellent resource for sexually interested and/or active adults over the age of 60. The book combines the author's personal reflections, questions and stories shared by older adults, and advice from sex therapists, sexuality educators, the author, and health care providers. The breadth of topics makes the book useful to…

  11. Using Visual Interpretive Analysis in Teaching Joyce Carol Oates's "Naked."

    ERIC Educational Resources Information Center

    Brandt, Kenneth K.

    2003-01-01

    Shares some of the classroom practices that have worked well in teaching "Naked." Presents a summary of introductory comments about the story and a description of the sketching activity. Offers interpretive commentary on a selection of representative student sketches that reflect significant features of the story. Provides general comments to…

  12. Registration of a tufted-naked seed upland cotton germplasm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A tufted-naked cotton (Gossypium hirsutum L.) mutant, 9023n4t (Reg. No. GP-971, PI 667553) was developed from the cultivar SC 9023 (9023) (PI 590933) through chemical mutagenesis. Germplasm line 9023n4t was developed by the Department of Plant and Soil Science, Texas Tech University, and released in...

  13. Back to Basics: Naked-Eye Astronomical Observation

    ERIC Educational Resources Information Center

    Barclay, Charles

    2003-01-01

    For pupils of both sexes and all ages from about six upwards, the subject of Astronomy holds many fascinations--the rapid changes in knowledge, the large resource of available IT packages and above all the beautiful pictures from Hubble and the large Earth-based telescopes. This article, however, stresses the excitement and importance of naked-eye…

  14. Two-Laser Interference Visible to the Naked Eye

    ERIC Educational Resources Information Center

    Kawalec, Tomasz; Bartoszek-Bober, Dobroslawa

    2012-01-01

    An experimental setup allowing the observation of two-laser interference by the naked eye is described. The key concept is the use of an electronic phase lock between two external cavity diode lasers. The experiment is suitable both for undergraduate and graduate students, mainly in atomic physics laboratories. It gives an opportunity for…

  15. Examination of poliovirus vaccine preparations for SV40 sequences.

    PubMed

    Sangar, D; Pipkin, P A; Wood, D J; Minor, P D

    1999-03-01

    Oral poliovaccines derived from the strains developed by Sabin have been the basis of vaccination against poliomyelitis in the U.K. since 1962. Contamination of earlier materials with the monkey virus SV40, particularly inactivated Salk type poliovaccines, is well documented. Precautions have been in place for more than 30 years to prevent SV40 contamination of oral poliovaccines based on screening of donor animals and tests for SV40 infectivity. PCR was applied to examine all archived samples of oral poliovaccines available to us dating from 1966 to the present, including all vaccines used in the U.K. since 1980, for the presence of SV40 sequences. Of 132 materials examined, 118 were negative on initial testing and fourteen gave reactions which on further examination were attributed either to cross contamination during handling in the laboratory at National Institute for Biological Standards and Control (NIBSC) or to non-specific amplification. It was concluded that none of the samples contained SV40 sequences. The materials included 69 separate monovalent bulks of poliovirus type 1, 2 or 3 grown on monkey kidney cells from four different manufacturers and 74 bulks grown on human diploid cells from two manufacturers. One additional seed material from 1962 contained low levels of unique and characteristic SV40 sequences. The seed had been treated by the manufacturer to inactivate DNA viruses and tests by the manufacturer and at NIBSC failed to demonstrate the presence of infectious SV40 virus. Monovalent bulks prepared by the manufacturer from this seed were negative for SV40 sequences by PCR. The PCR studies provide no evidence of contamination of oral poliovaccines used in the UK with infectious SV40 and suggest that the steps taken to ensure the absence of infectious SV40 are satisfactory. PMID:10441397

  16. Septage treatments to reduce the numbers of bacteria and polioviruses.

    PubMed Central

    Stramer, S L; Cliver, D O

    1984-01-01

    Disposal of the pumped contents of septic tanks (septage) represents a possible means of dissemination of enteric pathogens including viruses, since persistence of enteroviruses in septic tank sludge for greater than 100 days has been demonstrated. The risk of exposure to potentially infectious agents can be reduced by disinfecting septages before their disposal. Of the septage disinfectants examined (technical and analytical grade glutaraldehyde, hydrogen peroxide, heat treatments, and a combination of heat and hydrogen peroxide), the treatment including hydrogen peroxide (5 mg, plus 0.33 mg of trichloroacetic acid, per ml of septage) and 55 degrees C killed virtually all the bacteria in septage within 1 h, whereas 55 degrees C alone inactivated inoculated polioviruses within 30 min. Virus was the most sensitive to heat, whereas fecal coliforms appeared to be the most sensitive to all chemical treatments. The responses of fecal streptococci and virus to both grades of glutaraldehyde (each at 1 mg/ml) were similar. Virus was more resistant than either fecal streptococci or total bacteria to low concentrations of hydrogen peroxide (1 to 5 mg/ml); however, virus and fecal streptococci were more labile than total bacteria to the highest peroxide concentration (10 mg/ml) examined. It is possible that the treatment combining heat and hydrogen peroxide was the most effective in reducing the concentrations of all bacteria, because catalase and peroxidases as well as other enzymes were heat inactivated, although catalase seems the most likely cause of damage. However, this most effective treatment does not appear to be practical for on-site use as performed, so further work on septage disinfection is recommended. PMID:6093691

  17. Perfect fluid tori orbiting Kehagias-Sfetsos naked singularities

    NASA Astrophysics Data System (ADS)

    Stuchlík, Z.; Pugliese, D.; Schee, J.; Kučáková, H.

    2015-09-01

    We construct perfect fluid tori in the field of the Kehagias-Sfetsos (K-S) naked singularities. These are spherically symmetric vacuum solutions of the modified Hořava quantum gravity, characterized by a dimensionless parameter ω M^2, combining the gravitational mass parameter M of the spacetime with the Hořava parameter ω reflecting the role of the quantum corrections. In dependence on the value of ω M^2, the K-S naked singularities demonstrate a variety of qualitatively different behavior of their circular geodesics that is fully reflected in the properties of the toroidal structures, demonstrating clear distinction to the properties of the torii in the Schwarzschild spacetimes. In all of the K-S naked singularity spacetimes the tori are located above an "antigravity" sphere where matter can stay in a stable equilibrium position, which is relevant for the stability of the orbiting fluid toroidal accretion structures. The signature of the K-S naked singularity is given by the properties of marginally stable tori orbiting with the uniform distribution of the specific angular momentum of the fluid, l= const. In the K-S naked singularity spacetimes with ω M^2 > 0.2811, doubled tori with the same l= const can exist; mass transfer between the outer torus and the inner one is possible under appropriate conditions, while only outflow to the outer space is allowed in complementary conditions. In the K-S spacetimes with ω M^2 < 0.2811, accretion from cusped perfect fluid tori is not possible due to the non-existence of unstable circular geodesics.

  18. Naked bodies, naked genomes: the special (but not exceptional) nature of genomic information.

    PubMed

    Sulmasy, Daniel P

    2015-05-01

    Genetic exceptionalism, the view that genomic information is different from other types of sensitive information and deserves exceptional types of protections, has been roundly criticized. However, the public still expresses special fears about the access others might have to their genomic information. In this article, it is argued that there may be a basis for the public perception that genomic information is special, even if it cannot be said that policies could or should be enacted to protect the privacy and confidentiality of genomic information that would be exceptional relative to the protections one would enact to protect other types of sensitive information. The special nature of genomic information lies in understanding that it is neither personal property nor mere information. A genome is, at one and the same time, a physical aspect of a person and information about that person. Genomic data are embodied information that partially constitutes as well as describes individuals and that connects them in physical ways to their ancestors and their relatives. All forms of privacy need to be protected, but some intimate aspects of our lives command special respect. To see a genome is more analogous to seeing a naked body than to seeing a social security number. This metaphor suggests that clinicians and investigators ought to respect the special concerns of patients regarding genomic information while not claiming that there are any exceptional measures one could take to protect genomic privacy. Suggestions are given for how this view might affect patient interactions, consent discussions, public policy, and public trust in genomic research and clinical genetics. PMID:25232853

  19. Synthesis of immunogenic, but non-infectious, poliovirus particles in insect cells by a baculovirus expression vector.

    PubMed

    Urakawa, T; Ferguson, M; Minor, P D; Cooper, J; Sullivan, M; Almond, J W; Bishop, D H

    1989-06-01

    A baculovirus expression vector (AcLeon) derived from Autographa californica nuclear polyhedrosis virus (AcNPV) was prepared containing the complete 6.6 kb coding region of the P3/Leon/37 strain of poliovirus type 3 placed under the control of the AcNPV polyhedrin promoter. The recombinant virus was used to infect Spodoptera frugiperda insect cells. As demonstrated by use of the appropriate antibodies, infected insect cells made poliovirus proteins that included the structural proteins VP0, VP1 and VP3. Poliovirus particles were recovered from extracts of the infected cells and demonstrated to be free from detectable levels of RNA and to be non-infectious in tissue culture. After particle purification by CsCl gradient centrifugation and immunization of outbred mice, antibodies to the structural proteins, including neutralizing antibodies, were obtained. Other recombinant baculoviruses, containing the majority of the capsid coding region of P3/Leon/37 (e.g. AcCAP21, nucleotide residues 742 to 3318), made an unprocessed precursor to the poliovirus structural proteins. These data suggested that processing of the poliovirus gene product by the AcLeon construct was catalysed by the poliovirus-encoded proteases. The results demonstrated that antigenic and immunogenic poliovirus proteins and empty particles can be made in insect cells by recombinant baculoviruses. PMID:2543785

  20. Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

    PubMed Central

    van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

    2015-01-01

    Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

  1. Differential depuration of poliovirus, Escherichia coli, and a coliphage by the common mussel, Mytilus edulis

    SciTech Connect

    Power, U.F.; Collins, J.K. )

    1989-06-01

    The elimination of sewage effluent-associated poliovirus, Escherichia coli, and a 22-nm icosahedral coliphage by the common mussel, Mytilus edulis, was studied. Both laboratory-and commercial-scale recirculating, UV depuration systems were used in this study. In the laboratory system, the logarithms of the poliovirus, E. coli, and coliphage levels were reduced by 1.86, 2.9, and 2.16, respectively, within 52 h of depuration. The relative patterns and rates of elimination of the three organisms suggest that they are eliminated from mussels by different mechanisms during depuration under suitable conditions. Poliovirus was not included in experiments undertaken in the commercial-scale depuration system. The differences in the relative rates and patterns of elimination were maintained for E. coli and coliphage in this system, with the logarithm of the E. coli levels being reduced by 3.18 and the logarithm of the coliphage levels being reduced by 0.87. The results from both depuration systems suggest that E. coli is an inappropriate indicator of the efficiency of virus elimination during depuration. The coliphage used appears to be a more representative indicator. Depuration under stressful conditions appeared to have a negligible affect on poliovirus and coliphage elimination rates from mussels. However, the rate and pattern of E. coli elimination were dramatically affected by these conditions. Therefore, monitoring E. coli counts might prove useful in ensuring that mussels are functioning well during depuration.

  2. CONCENTRATION OF POLIOVIRUS FROM TAP WATER USING POSITIVELY CHARGED MICROPOROUS FILTERS

    EPA Science Inventory

    Microporous filters that are more electropositive than the negatively charged filters currently used for virus concentration from water by filter adsorption-elution methods were evaluated for poliovirus recovery from tap water. Zeta Plus filters composed of diatomaceous earth-cel...

  3. Concentration of poliovirus from tap water using positively charged microporous filters.

    PubMed Central

    Sobsey, M D; Jones, B L

    1979-01-01

    Microporous filters that are more electropositive than the negatively charged filters currently used for virus concentrations from water by filter adsorption-elution methods were evaluated for poliovirus recovery from tap water. Zeta Plus filters composed of diatomaceous earth-cellulose-"charge-modified" resin mixtures and having a net positive charge of up to pH 5 to 6 efficiently adsorbed poliovirus from tap water at ambient pH levels 7.0 to 7.5 without added multivalent cation salts. The adsorbed virus were eluted with glycine-NaOH, pH 9.5 to 11.5. Electropositive asbestos-cellulose filters efficiently adsorbed poliovirus from tap water without added multivalent cation salts between pH 3.5 and 9.0, and the absorbed viruses could be eluted with 3% beef extract, pH 9, but not with pH 9.5 to 11.5 glycine-NaOH. Under water quality conditions in which poliovirus recoveries from large volumes of water were less than 5% with conventional negatively charged filters and standard methods, recoveries with Zeta Plus filters averaged 64 and 22.5% for one- and two-stage concentration procedures, respectively. Electropositive filters appear to offer distinct advantages over conventional negatively charged filters for concentrating enteric viruses from water, and their behavior tends to confirm the importance of electrostatic forces in virus recovery from water by microporous filter adsorption-elution methods. PMID:36844

  4. Update on Vaccine-Derived Polioviruses - Worldwide, January 2015-May 2016.

    PubMed

    Jorba, Jaume; Diop, Ousmane M; Iber, Jane; Sutter, Roland W; Wassilak, Steven G; Burns, Cara C

    2016-01-01

    In 1988, the World Health Assembly resolved to eradicate poliomyelitis worldwide (1). One of the main tools used in polio eradication efforts has been the live, attenuated, oral poliovirus vaccine (OPV) (2), an inexpensive vaccine easily administered by trained volunteers. OPV might require several doses to induce immunity, but provides long-term protection against paralytic disease. Through effective use of OPV, the Global Polio Eradication Initiative (GPEI) has brought wild polioviruses to the threshold of eradication (1). However, OPV use, particularly in areas with low routine vaccination coverage, is associated with the emergence of genetically divergent vaccine-derived polioviruses (VDPVs) whose genetic drift from the parental OPV strains indicates prolonged replication or circulation (3). VDPVs can emerge among immunologically normal vaccine recipients and their contacts as well as among persons with primary immunodeficiencies (PIDs). Immunodeficiency-associated VDPVs (iVDPVs) can replicate for years in some persons with PIDs. In addition, circulating vaccine-derived polioviruses (cVDPVs) (3) can emerge in areas with low OPV coverage and can cause outbreaks of paralytic polio. This report updates previous summaries regarding VDPVs (4). PMID:27491079

  5. EFFECTS OF BENTONITE CLAY SOLIDS ON POLIOVIRUS CONCENTRATION FROM WATER BY MICROPOROUS FILTER METHODS

    EPA Science Inventory

    In order to determine if suspended solids interfere with enteric virus recovery from water by microporous filter methods, the effects of bentonite clay solids at a concentration of 10 NTU on the recovery of poliovirus type 1 from seeded, activated carbon-treated, filtered tap wat...

  6. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    SciTech Connect

    Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

    2011-10-14

    Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

  7. Innovative IPV from attenuated Sabin poliovirus or newly designed alternative seed strains.

    PubMed

    Hamidi, Ahd; Bakker, Wilfried A M

    2012-11-01

    This article gives an overview of the patent literature related to innovative inactivated polio vaccine (i-IPV) based on using Sabin poliovirus strains and newly developed alternative recombinant poliovirus strains. This innovative approach for IPV manufacturing is considered to attribute to the requirement for affordable IPV in the post-polio-eradication era, which is on the horizon. Although IPV is a well-established vaccine, the number of patent applications in this field was seen to have significantly increased in the past decade. Currently, regular IPV appears to be too expensive for universal use. Future affordability may be achieved by using alternative cell lines, alternative virus seed strains, improved and optimized processes, dose sparing, or the use of adjuvants. A relatively short-term option to achieve cost-price reduction is to work on regular IPV, using wild-type poliovirus strains, or on Sabin-IPV, based on using attenuated poliovirus strains. This price reduction can be achieved by introducing efficiency in processing. There are also multiple opportunities to work on dose sparing, for example, by using adjuvants or fractional doses. Renewed interest in this field was clearly reflected in the number and diversity of patent applications. In a later stage, several innovative approaches may become even more attractive, for example the use of recombinant virus strains or even a totally synthetic vaccine. Currently, such work is mainly carried out by research institutes and universities and therefore clinical data are not available. PMID:24236927

  8. Capped mRNAs with reduced secondary structure can function in extracts from poliovirus-infected cells

    SciTech Connect

    Sonenberg, N.; Guertin, D.; Lee, K.A.W.

    1982-12-01

    Extracts form poliovirus-infected HeLa cells were used to study ribosome binding of native and denatured reovirus mRNAs and translation of capped mRNAs with different degrees of secondary structure. Here, the authors demonstrate that ribosomes in extracts from poliovirus-infected cells could form initiation complexes with denatured reovirus mRNA, in contrast to their inability to bind native reovirus mRNA. Furthermore, the capped alfalfa mosiac virus 4 RNA, which is most probable devoid of stable secondary structure at its 5' end, could be translated at much higher efficiency than could other capped mRNAs in extracts from poliovirus-infected cells.

  9. Structural studies of human Naked2: A biologically active intrinsically unstructured protein

    SciTech Connect

    Hu Tianhui; Krezel, Andrzej M.; Li Cunxi; Coffey, Robert J. . E-mail: robert.coffey@vanderbilt.edu

    2006-12-01

    Naked1 and 2 are two mammalian orthologs of Naked Cuticle, a canonical Wnt signaling antagonist in Drosophila. Naked2, but not Naked1, interacts with transforming growth factor-{alpha} (TGF{alpha}) and escorts TGF{alpha}-containing vesicles to the basolateral membrane of polarized epithelial cells. Full-length Naked2 is poorly soluble. Since most functional domains, including the Dishevelled binding region, EF-hand, vesicle recognition, and membrane targeting motifs, reside in the N-terminal half of the protein, we expressed and purified the first 217 residues of human Naked2 and performed a functional analysis of this fragment. Its circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra showed no evidence of secondary and/or tertiary structure. The fragment did not bind calcium or zinc. These results indicate that the N-terminal half of Naked2 behaves as an intrinsically unstructured protein.

  10. Update on vaccine-derived polioviruses - worldwide, July 2012-December 2013.

    PubMed

    Diop, Ousmane M; Burns, Cara C; Wassilak, Steven G; Kew, Olen M

    2014-03-21

    In 1988, the World Health Assembly resolved to eradicate poliomyelitis worldwide. One of the main tools used in polio eradication efforts has been live, attenuated oral poliovirus vaccine (OPV), an inexpensive vaccine easily administered by trained volunteers. OPV might require several doses to induce immunity, but then it provides long-term protection against paralytic disease through durable humoral immunity. Rare cases of vaccine-associated paralytic poliomyelitis can occur among immunologically normal OPV recipients, their contacts, and persons who are immunodeficient. In addition, vaccine-derived polioviruses (VDPVs) can emerge in areas with low OPV coverage to cause polio outbreaks and can replicate for years in persons who have primary, B-cell immunodeficiencies. This report updates previous surveillance summaries and describes VDPVs detected worldwide during July 2012-December 2013. Those include a new circulating VDPV (cVDPV) outbreak identified in Pakistan in 2012, with spread to Afghanistan; an outbreak in Afghanistan previously identified in 2009 that continued into 2013; a new outbreak in Chad that spread to Cameroon, Niger, and northeastern Nigeria; and an outbreak that began in Somalia in 2008 that continued and spread to Kenya in 2013. A large outbreak in Nigeria that was identified in 2005 was nearly stopped by the end of 2013. Additionally, 10 newly identified persons in eight countries were found to excrete immunodeficiency-associated VDPVs (iVDPVs), and VDPVs were found among immunocompetent persons and environmental samples in 13 countries. Because the majority of VDPV isolates are type 2, the World Health Organization has developed a plan for coordinated worldwide replacement of trivalent OPV (tOPV) with bivalent OPV (bOPV; types 1 and 3) by 2016, preceded by introduction of at least 1 dose of inactivated poliovirus vaccine (IPV) containing all three poliovirus serotypes into routine immunization schedules worldwide to ensure high population

  11. Naked-eye nanobiosensor for therapeutic drug monitoring of methotrexate.

    PubMed

    Yockell-Lelièvre, H; Bukar, N; Toulouse, J L; Pelletier, J N; Masson, J-F

    2016-01-21

    Sensing of methotrexate at clinically-relevant concentrations was achieved with a plasmon-coupling assay. In this assay, free methotrexate and folic acid Au nanoparticles competed for human dihydrofolate reductase (hDHFR)-functionalized Au nanoparticles (Au NP). The hDHFR-functionalized Au NPs were immobilized on a small glass sensor inserted in a portable 4-channel LSPR reader. This allowed rapid (minutes) and sensitive (nanomolar range) measurement of methotrexate concentration by means of total internal reflection plasmonic spectroscopy. The large bathochromic shifts of the plasmon-coupling assay led to striking colour changes visible to the naked eye for methotrexate at clinically-relevant concentrations. The results demonstrate the potential for therapeutic drug monitoring of a widely used chemotherapy agent, as assessed with the naked eye. PMID:26229988

  12. Why do naked singularities form in gravitational collapse? II

    SciTech Connect

    Joshi, Pankaj S.; Goswami, Rituparno; Dadhich, Naresh

    2004-10-15

    We examine physical features that could lead to formation of a naked singularity rather than black hole, as end state of spherical collapse. Generalizing earlier results on dust collapse to general type I matter fields, it is shown that collapse always creates black hole if shear vanishes or density is homogeneous. It follows that nonzero shear is a necessary condition for singularity to be visible to external observers, when trapped surface formation is delayed by shearing forces or inhomogeneity within the collapsing cloud.

  13. Naked-eye acquisition of visible near-earth satellites

    NASA Technical Reports Server (NTRS)

    Mansfield, Roger L.

    1988-01-01

    This paper develops visual sighting criteria for observing artificial near-earth satellites with the naked eye, and summarizes the mathematics needed to predict visibility efficiently from a set of mean orbital elements. It reports on a successful application of the sighting criteria and visibility-prediction mathematics, and shows that successful visual acquisition depends as much upon the observer's skill in recognizing the constellations as it does upon accurate visibility predictions.

  14. Virus replicon particle based Chikungunya virus neutralization assay using Gaussia luciferase as readout

    PubMed Central

    2013-01-01

    Background Chikungunya virus (CHIKV) has been responsible for large epidemic outbreaks causing fever, headache, rash and severe arthralgia. So far, no specific treatment or vaccine is available. As nucleic acid amplification can only be used during the viremic phase of the disease, serological tests like neutralization assays are necessary for CHIKV diagnosis and for determination of the immune status of a patient. Furthermore, neutralization assays represent a useful tool to validate the efficacy of potential vaccines. As CHIKV is a BSL3 agent, neutralization assays with infectious virus need to be performed under BSL3 conditions. Our aim was to develop a neutralization assay based on non-infectious virus replicon particles (VRPs). Methods VRPs were produced by cotransfecting baby hamster kidney-21 cells with a CHIKV replicon expressing Gaussia luciferase (Gluc) and two helper RNAs expressing the CHIKV capsid protein or the remaining structural proteins, respectively. The resulting single round infectious particles were used in CHIKV neutralization assays using secreted Gluc as readout. Results Upon cotransfection of a CHIKV replicon expressing Gluc and the helper RNAs VRPs could be produced efficiently under optimized conditions at 32°C. Infection with VRPs could be measured via Gluc secreted into the supernatant. The successful use of VRPs in CHIKV neutralization assays was demonstrated using a CHIKV neutralizing monoclonal antibody or sera from CHIKV infected patients. Comparison of VRP based neutralization assays in 24- versus 96-well format using different amounts of VRPs revealed that in the 96-well format a high multiplicity of infection is favored, while in the 24-well format reliable results are also obtained using lower infection rates. Comparison of different readout times revealed that evaluation of the neutralization assay is already possible at the same day of infection. Conclusions A VRP based CHIKV neutralization assay using Gluc as readout

  15. Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia

    PubMed Central

    2014-01-01

    Background Rhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids. Results Genome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones. Conclusion Variable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. The set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization

  16. PER.C6(®) cells as a serum-free suspension cell platform for the production of high titer poliovirus: a potential low cost of goods option for world supply of inactivated poliovirus vaccine.

    PubMed

    Sanders, Barbara P; Edo-Matas, Diana; Custers, Jerome H H V; Koldijk, Martin H; Klaren, Vincent; Turk, Marije; Luitjens, Alfred; Bakker, Wilfried A M; Uytdehaag, Fons; Goudsmit, Jaap; Lewis, John A; Schuitemaker, Hanneke

    2013-01-21

    There are two highly efficacious poliovirus vaccines: Sabin's live-attenuated oral polio vaccine (OPV) and Salk's inactivated polio vaccine (IPV). OPV can be made at low costs per dose and is easily administrated. However, the major drawback is the frequent reversion of the OPV vaccine strains to virulent poliovirus strains which can result in Vaccine Associated Paralytic Poliomyelitis (VAPP) in vaccinees. Furthermore, some OPV revertants with high transmissibility can circulate in the population as circulating Vaccine Derived Polioviruses (cVDPVs). IPV does not convey VAPP and cVDPVs but the high costs per dose and insufficient supply have rendered IPV an unfavorable option for low and middle-income countries. Here, we explored whether the human PER.C6(®) cell-line, which has the unique capability to grow at high density in suspension, under serum-free conditions, could be used as a platform for high yield production of poliovirus. PER.C6(®) cells supported replication of all three poliovirus serotypes with virus titers ranging from 9.4 log(10) to 11.1 log(10)TCID(50)/ml irrespective of the volume scale (10 ml in shaker flasks to 2 L in bioreactors). This production yield was 10-30 fold higher than in Vero cell cultures performed here, and even 100-fold higher than what has been reported for Vero cell cultures in literature [38]. In agreement, the D-antigen content per volume PER.C6(®)-derived poliovirus was on average 30-fold higher than Vero-derived poliovirus. Interestingly, PER.C6(®) cells produced on average 2.5-fold more D-antigen units per cell than Vero cells. Based on our findings, we are exploring PER.C6(®) as an interesting platform for large-scale production of poliovirus at low costs, potentially providing the basis for global supply of an affordable IPV. PMID:23123018

  17. Effects of hydrostatic pressure on the stability and thermostability of poliovirus: a new method for vaccine preservation.

    PubMed

    Ferreira, Evanilce; Mendes, Ygara S; Silva, Jerson L; Galler, Ricardo; Oliveira, Andréa C; Freire, Marcos S; Gaspar, Luciane P

    2009-08-27

    Viruses are a structurally diverse group of infectious agents that differ widely in their sensitivities to high hydrostatic pressure (HHP). Studies on picornaviruses have demonstrated that these viruses are extremely resistant to HHP treatments, with poliovirus appearing to be the most resistant. Here, the three attenuated poliovirus serotypes were compared with regard to pressure and thermal resistance. We found that HHP does not inactivate any of the three serotypes studied (1-3). Rather, HHP treatment was found to stabilize poliovirus by increasing viral thermal resistance at 37 degrees C. Identification of new methods that stabilize poliovirus against heat inactivation would aid in the design of a more heat-stable vaccine, circumventing the problems associated with refrigeration during storage and transport of the vaccine prior to use. PMID:19616496

  18. Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV) in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating...

  19. Development of dengue virus replicons expressing HIV-1 gp120 and other heterologous genes: a potential future tool for dual vaccination against dengue virus and HIV

    PubMed Central

    Pang, Xiaowu; Zhang, Mingjie; Dayton, Andrew I

    2001-01-01

    Background Toward the goals of providing an additional vector to add to the armamentarium available to HIV vaccinologists and of creating a bivalent vaccine effective against dengue virus and HIV, we have attempted to create vectors which express dengue virus non-structural proteins and HIV immunogens. Previously we reported the successful construction of dengue virus replicons which lack structural genes necessary for virion release and spreading infection in culture but which can replicate intracellularly and abundantly produce dengue non-structural proteins. Here we attempted to express heterologous genetic material from these replicons. Results We cloned into a Δpre-M/E dengue virus replicon genes for either green fluorescent protein (GFP), HIV gp160 or HIV gp120 and tested the ability of these constructs to express dengue virus proteins as well as the heterologous proteins in tissue culture after transfection of replicon RNA. Conclusions Heterologous proteins were readily expressed from these constructs. GFP and gp120 demonstrated minimal or no toxicity. Gp160 expressing replicons were found to express proteins abundantly at 36 hours post transfection, but after 50 hrs of transfection, few replicon positive cells could be found despite the presence of cellular debris positive for replicon proteins. This suggested that gp160 expressed from dengue virus replicons is considerably more toxic than either GFP or gp120. The successful expression of heterologous proteins, including HIV gp120 for long periods in culture suggests this vector system may be useful as a vaccine vector, given appropriate delivery methods. PMID:11747468

  20. Lack of interference with immunogenicity of a chimeric alphavirus replicon particle-based influenza vaccine by preexisting antivector immunity.

    PubMed

    Uematsu, Yasushi; Vajdy, Michael; Lian, Ying; Perri, Silvia; Greer, Catherine E; Legg, Harold S; Galli, Grazia; Saletti, Giulietta; Otten, Gillis R; Rappuoli, Rino; Barnett, Susan W; Polo, John M

    2012-07-01

    Antivector immunity has been recognized as a potential caveat of using virus-based vaccines. In the present study, an alphavirus-based replicon particle vaccine platform, which has demonstrated robust immunogenicity in animal models, was tested for effects of antivector immunity on immunogenicity against hemagglutinin of influenza virus as a target antigen and efficacy for protection against lethal challenge with the virus. Chimeric alphavirus-based replicon particles, comprising Venezuelan equine encephalitis virus nonstructural and Sindbis virus structural components, induced efficient protective antibody responses, which were not adversely influenced after multiple immunizations with the same vector expressing various antigens. PMID:22623651

  1. Nectin-Like Interactions between Poliovirus and Its Receptor Trigger Conformational Changes Associated with Cell Entry

    PubMed Central

    Strauss, Mike; Filman, David J.; Belnap, David M.; Cheng, Naiqian; Noel, Roane T.

    2015-01-01

    ABSTRACT Poliovirus infection is initiated by attachment to a receptor on the cell surface called Pvr or CD155. At physiological temperatures, the receptor catalyzes an irreversible expansion of the virus to form an expanded form of the capsid called the 135S particle. This expansion results in the externalization of the myristoylated capsid protein VP4 and the N-terminal extension of the capsid protein VP1, both of which become inserted into the cell membrane. Structures of the expanded forms of poliovirus and of several related viruses have recently been reported. However, until now, it has been unclear how receptor binding triggers viral expansion at physiological temperature. Here, we report poliovirus in complex with an enzymatically partially deglycosylated form of the 3-domain ectodomain of Pvr at a 4-Å resolution, as determined by cryo-electron microscopy. The interaction of the receptor with the virus in this structure is reminiscent of the interactions of Pvr with its natural ligands. At a low temperature, the receptor induces very few changes in the structure of the virus, with the largest changes occurring within the footprint of the receptor, and in a loop of the internal protein VP4. Changes in the vicinity of the receptor include the displacement of a natural lipid ligand (called “pocket factor”), demonstrating that the loss of this ligand, alone, is not sufficient to induce particle expansion. Finally, analogies with naturally occurring ligand binding in the nectin family suggest which specific structural rearrangements in the virus-receptor complex could help to trigger the irreversible expansion of the capsid. IMPORTANCE The cell-surface receptor (Pvr) catalyzes a large structural change in the virus that exposes membrane-binding protein chains. We fitted known atomic models of the virus and Pvr into three-dimensional experimental maps of the receptor-virus complex. The molecular interactions we see between poliovirus and its receptor are

  2. Improved Genotyping Vaccine and Wild-Type Poliovirus Strains by Restriction Fragment Length Polymorphism Analysis: Clinical Diagnostic Implications

    PubMed Central

    Georgopoulou, Amalia; Markoulatos, Panayotis; Spyrou, Niki; Vamvakopoulos, Nicholas C.

    2000-01-01

    The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5′ noncoding region of polioviruses was selected for RT-PCR amplification by the UC53-UG52 primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, and AvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, or NcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success. PMID:11101561

  3. Improved genotyping vaccine and wild-type poliovirus strains by restriction fragment length polymorphism analysis: clinical diagnostic implications.

    PubMed

    Georgopoulou, A; Markoulatos, P; Spyrou, N; Vamvakopoulos, N C

    2000-12-01

    The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5' noncoding region of polioviruses was selected for RT-PCR amplification by the UC(53)-UG(52) primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, and AvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, or NcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success. PMID:11101561

  4. Is it really naked? On cosmic censorship in string theory

    SciTech Connect

    Frolov, Andrei V.

    2004-11-15

    We investigate the possibility of cosmic censorship violation in string theory using a characteristic double-null code, which penetrates horizons and is capable of resolving the spacetime all the way to the singularity. We perform high-resolution numerical simulations of the evolution of negative mass initial scalar field profiles, which were argued to provide a counterexample to cosmic censorship conjecture for AdS-asymptotic spacetimes in five-dimensional supergravity. In no instances formation of naked singularity is seen. Instead, numerical evidence indicates that black holes form in the collapse. Our results are consistent with earlier numerical studies, and explicitly show where the 'no black hole' argument breaks.

  5. Is it Really Naked? On Cosmic Censorship in String Theory

    SciTech Connect

    Frolov, A

    2004-09-30

    We investigate the possibility of cosmic censorship violation in string theory using a characteristic double-null code, which penetrates horizons and is capable of resolving the spacetime all the way to the singularity. We perform high-resolution numerical simulations of the evolution of negative mass initial scalar field profiles, which were argued to provide a counter example to cosmic censorship conjecture for AdS-asymptotic spacetimes in five-dimensional supergravity. In no instances formation of naked singularity is seen. Instead, numerical evidence indicates that black holes form in the collapse. Our results are consistent with earlier numerical studies, and explicitly show where the ''no black hole'' argument breaks.

  6. Luminous variable stars with naked eye: data reduction including extinction

    NASA Astrophysics Data System (ADS)

    Sigismondi, Costantino

    2014-05-01

    The observation of variable stars from urban contexts is hampered by city lights and field of view. Some bright stars like Betelgeuse and Antares are visible from the majority of cities, and during clear nights can be accurately estimated with the naked eye. The reference stars should be bright stars, not necessarily at the same altitude, including the atmospheric extinction in the data reduction. The software Stellarium 0.12.4 calculates well the standard atmospheric extinction with stars at least 10 degrees above the horizon. The accuracy of visual estimations is better than 0.1 magnitudes.

  7. Notes from the Field: Circulating Vaccine-Derived Poliovirus Outbreaks - Five Countries, 2014-2015.

    PubMed

    Morales, Michelle; Nnadi, Chimeremma D; Tangermann, Rudolf H; Wassilak, Steven G F

    2016-02-12

    In 2015, wild poliovirus (WPV) transmission was identified in only Afghanistan and Pakistan (1). The widespread use of live, attenuated oral poliovirus vaccine (OPV) has been key in polio eradication efforts. However, OPV use, particularly in areas with low vaccination coverage, is associated with the low risk for emergence of vaccine-derived polioviruses (VDPV), which can cause paralysis (2). VDPVs vary genetically from vaccine viruses and can cause outbreaks in areas with low vaccination coverage. Circulating VDPVs (cVDPVs) are VDPVs in confirmed outbreaks. Single VDPVs for which the origin cannot be determined are classified as ambiguous (aVDPVs), which can also cause paralysis. Among the three types of WPV, type 2 has been declared to be eradicated. More than 90% of cVDPV cases have been caused by type 2 cVDPVs (cVDPV2). Therefore, in April 2016, all OPV-using countries of the world are discontinuing use of type 2 Sabin vaccine by simultaneously switching from trivalent OPV (types 1, 2, and 3) to bivalent OPV (types 1 and 3) for routine and supplementary immunization. The World Health Organization recently broadened the definition of cVDPVs to include any VDPV with genetic evidence of prolonged transmission (i.e., >1.5 years) and indicated that any single VDPV2 event (a case of paralysis caused by a VDPV or isolation of a VDPV from an environmental specimen) should elicit a detailed outbreak investigation and local immunization response. A confirmed cVDPV2 detection should elicit a full poliovirus outbreak response that includes multiple supplemental immunization activities (SIAs); an aVDPV designation should be made only after investigation and response (3). Since 2005, there have been 1-8 cVDPV outbreaks and 3-12 aVDPV events per year. There are currently five active cVDPV outbreaks in Guinea, Laos, Madagascar, Myanmar, and Ukraine, and four other active VDPV events. PMID:26866942

  8. The poliovirus receptor protein is produced both as membrane-bound and secreted forms.

    PubMed Central

    Koike, S; Horie, H; Ise, I; Okitsu, A; Yoshida, M; Iizuka, N; Takeuchi, K; Takegami, T; Nomoto, A

    1990-01-01

    Both genomic and complementary DNA clones encoding poliovirus receptors were isolated from genomic and complementary DNA libraries prepared from HeLa S3 cells, respectively. Nucleotide sequence analysis of these cloned DNAs revealed that the poliovirus receptor gene is approximately 20 kb long and contains seven introns in the coding region, and that at least four mRNA isoforms referring to the coding sequence are generated by alternative splicing and appear to encode four different molecules, that is, PVR alpha, PVR beta, PVR gamma and PVR delta. The predicted amino acid sequences indicate that PVR alpha and PVR delta, corresponding to the previously described cDNA clones H20A and H20B, respectively, are integral membrane proteins while the other two molecules described here for the first time lack a putative transmembrane domain. Mouse cell transformants carrying PVR alpha were permissive for poliovirus infection, but those carrying PVR beta were hardly permissive. In contrast to PVR alpha, PVR beta was not detected on the surface of the mouse cell transformants but was detected in the culture fluid by an immunological method using a monoclonal antibody against poliovirus receptor. Three types of splicing products for PVR alpha, PVR beta and PVR gamma were detected by polymerase chain reactions using appropriate primers in poly(A)+ RNAs of the brain, leukocyte, liver, lung and placenta of humans; the choice of primers used did not permit detection of PVR delta. In situ hybridization using a cDNA fragment as a probe demonstrated that the PVR gene is located at the band q13.1----13.2 of human chromosome 19. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2170108

  9. Time delay and magnification centroid due to gravitational lensing by black holes and naked singularities

    SciTech Connect

    Virbhadra, K. S.; Keeton, C. R.

    2008-06-15

    We model the massive dark object at the center of the Galaxy as a Schwarzschild black hole as well as Janis-Newman-Winicour naked singularities, characterized by the mass and scalar charge parameters, and study gravitational lensing (particularly time delay, magnification centroid, and total magnification) by them. We find that the lensing features are qualitatively similar (though quantitatively different) for Schwarzschild black holes, weakly naked, and marginally strongly naked singularities. However, the lensing characteristics of strongly naked singularities are qualitatively very different from those due to Schwarzschild black holes. The images produced by Schwarzschild black hole lenses and weakly naked and marginally strongly naked singularity lenses always have positive time delays. On the other hand, strongly naked singularity lenses can give rise to images with positive, zero, or negative time delays. In particular, for a large angular source position the direct image (the outermost image on the same side as the source) due to strongly naked singularity lensing always has a negative time delay. We also found that the scalar field decreases the time delay and increases the total magnification of images; this result could have important implications for cosmology. As the Janis-Newman-Winicour metric also describes the exterior gravitational field of a scalar star, naked singularities as well as scalar star lenses, if these exist in nature, will serve as more efficient cosmic telescopes than regular gravitational lenses.

  10. Simian virus 40, poliovirus vaccines, and human cancer: research progress versus media and public interests

    NASA Technical Reports Server (NTRS)

    Butel, J. S.

    2000-01-01

    From 1955 through early 1963, millions of people were inadvertently exposed to simian virus 40 (SV40) as a contaminant of poliovirus vaccines; the virus had been present in the monkey kidney cultures used to prepare the vaccines and had escaped detection. SV40 was discovered in 1960 and subsequently eliminated from poliovirus vaccines. This article reviews current knowledge about SV40 and considers public responses to reports in the media. SV40 is a potent tumour virus with broad tissue tropism that induces tumours in rodents and transforms cultured cells from many species. It is also an important laboratory model for basic studies of molecular processes in eukaryotic cells and mechanisms of neoplastic transformation. SV40 neutralizing antibodies have been detected in individuals not exposed to contaminated poliovirus vaccines. There have been many reports of detection of SV40 DNA in human tumours, especially mesotheliomas, brain tumours and osteosarcomas; and DNA sequence analyses have ruled out the possibility that the viral DNA in tumours was due to laboratory contamination or that the virus had been misidentified. However, additional studies are necessary to prove that SV40 is the cause of certain human cancers. A recently published review article evaluated the status of the field and received much media attention. The public response emphasized that there is great interest in the possibility of health risks today from vaccinations received in the past.

  11. Strand-specific attachment of avidin-spheres to double-stranded poliovirus RNA.

    PubMed Central

    Richards, O C; Ehrenfeld, E; Manning, J

    1979-01-01

    Poliovirus-specific double-stranded RNA molecules containing covalently attached protein were coupled with a biotin ester through the protein moiety. Subsequent interaction of the RNA-biotin with avidin attached to electronopaque plastic spheres led to the formation of complexes that were easily visualized in the electron microscope. Avidinspheres were associated only with one end of the RNA-biotin molecules, as seen in the electron microscope. Avidin-sphere attachment to poliovirus double-stranded RNA is strand specific, as shown by molecular hybridization of strand-specific probes to the separated strands of denatured complexes. [3H]DNA complementary to polio virion RNA hybridized exclusively to the strands bearing associated spheres [(+) strands] whereas 125I-labeled virion RNA hybridized predominantly with strands without spheres [(-)strands]. This biotin-avidin labeling technique provides a means for the isolation of full-length poliovirus (-) strands and may provide a general means for isolation of double-stranded polynucleotides containing tightly attached protein. Images PMID:218216

  12. Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol.

    PubMed Central

    Neufeld, K L; Galarza, J M; Richards, O C; Summers, D F; Ehrenfeld, E

    1994-01-01

    A terminal adenylyl transferase (TATase) activity has been identified in preparations of purified poliovirus RNA-dependent RNA polymerase (3Dpol). Highly purified 3Dpol is capable of adding [32P]AMP to the 3' ends of chemically synthesized 12-nucleotide (nt)-long RNAs. The purified 52-kDa polypeptide, isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured, retained the TATase activity. Two 3Dpol mutants, purified from Escherichia coli expression systems, displayed no detectable polymerase activity and were unable to catalyze TATase activity. Likewise, extracts from the parental E. coli strain that harbored no expression plasmid were unable to catalyze formation of the TATase products. With the RNA oligonucleotide 5'-CCUGCUUUUGCA-3' used as an acceptor, the products formed by wild-type 3Dpol were 9 and 18 nt longer than the 12-nt oligomer. GTP, CTP, and UTP did not serve as substrates for transfer to this RNA, either by themselves or when all deoxynucleoside triphosphates were present in the reaction. Results from kinetic and stoichiometric analyses suggest that the reaction is catalytic and shows substrate and enzyme dependence. The 3'-terminal 13 nt of poliovirus minus-strand RNA also served as an acceptor for TATase activity, raising the possibility that this activity functions in poliovirus RNA replication. The efficiency of utilization and the nature of the products formed during the reaction were dependent on the acceptor RNA. Images PMID:8057462

  13. Recovery of poliovirus from cut surface of stored fresh papaya fruit.

    PubMed

    Lee, A S; Yap, K L

    1999-06-01

    Poliovirus kept on the cut surfaces of fully ripe papaya cubes placed in an ice box showed a sharp and significant reduction in the recovery of infectious virus about 15 minutes after exposure. Thereafter, a very gradual decrease ensued and infectious residual virus was detected up to the end of the 6-hour exposure period. Papaya cubes washed or kept overnight before virus inoculation, and from less ripe fruits produced a similar survival pattern. A very small proportion of the inoculum was recovered from the mashed content of the inoculated papaya cubes thus suggesting that most of the non-recovered virus particles were inactivated. The results suggest that the importance of poliovirus-contaminated cut papayas as a transmission vehicle for the virus is greatly reduced by the rapid decline in the infectivity of a large proportion of the virus soon after contamination. Nevertheless, the potential to transmit remains as a small residual pool of infectious poliovirus is able to survive for a relatively long period. PMID:10774695

  14. Massive outbreak of poliomyelitis caused by type-3 wild poliovirus in Angola in 1999.

    PubMed Central

    Valente, F.; Otten, M.; Balbina, F.; Van de Weerdt, R.; Chezzi, C.; Eriki, P.; Van-Dúnnen, J.; Bele, J. M.

    2000-01-01

    The largest outbreak of poliomyelitis ever recorded in Africa (1093 cases) occurred from 1 March to 28 May 1999 in Luanda, Angola, and in surrounding areas. The outbreak was caused primarily by a type-3 wild poliovirus, although type-1 wild poliovirus was circulating in the outbreak area at the same time. Infected individuals ranged in age from 2 months to 22 years; 788 individuals (72%) were younger than 3 years. Of the 590 individuals whose vaccination status was known, 23% had received no vaccine and 54% had received fewer than three doses of oral poliovirus vaccine (OPV). The major factors that contributed to this outbreak were as follows: massive displacement of unvaccinated persons to urban settings; low routine OPV coverage; inaccessible populations during the previous three national immunization days (NIDs); and inadequate sanitation. This outbreak indicates the urgent need to improve accessibility to all children during NIDs and the dramatic impact that war can have by displacing persons and impeding access to routine immunizations. The period immediately after an outbreak provides an enhanced opportunity to eradicate poliomyelitis. If continuous access in all districts for acute flaccid paralysis surveillance and supplemental immunizations cannot be assured, the current war in Angola may threaten global poliomyelitis eradication. PMID:10812730

  15. A Polyprotein-Expressing Salmonid Alphavirus Replicon Induces Modest Protection in Atlantic Salmon (Salmo Salar) Against Infectious Pancreatic Necrosis

    PubMed Central

    Abdullah, Azila; Olsen, Christel M.; Hodneland, Kjartan; Rimstad, Espen

    2015-01-01

    Vaccination is an important strategy for the control and prevention of infectious pancreatic necrosis (IPN) in farmed Atlantic salmon (Salmo salar) in the post-smolt stage in sea-water. In this study, a heterologous gene expression system, based on a replicon construct of salmonid alphavirus (SAV), was used for in vitro and in vivo expression of IPN virus proteins. The large open reading frame of segment A, encoding the polyprotein NH2-pVP2-VP4-VP3-COOH, as well as pVP2, were cloned and expressed by the SAV replicon in Chinook salmon embryo cells (CHSE-214) and epithelioma papulosum cyprini (EPC) cells. The replicon constructs pSAV/polyprotein (pSAV/PP) and pSAV/pVP2 were used to immunize Atlantic salmon (Salmo salar) by a single intramuscular injection and tested in a subsequent IPN virus (IPNV) challenge trial. A low to moderate protection against IPN was observed in fish immunized with the replicon vaccine that encoded the pSAV/PP, while the pSAV/pVP2 construct was not found to induce protection. PMID:25606973

  16. Development of porcine respiratory and reproductive syndrome virus replicon vector for foot-and-mouth disease vaccine

    PubMed Central

    Jeeva, Subbiah; Lee, Jung-Ah; Park, Seung-Yong; Song, Chang-Seon; Choi, In-Soo

    2014-01-01

    Purpose Foot-and-mouth disease (FMD) is an economically important global animal disease. To control FMD virus (FMDV) outbreaks, a lot of different novel approaches have been attempted. In this study, we proposed a novel porcine reproductive and respiratory syndrome virus (PRRSV) as a replicon vector to express FMDV structural protein. Materials and Methods PRRSV infectious clone (PRRSVK418DM) was used to develop an expression vector through the reverse genetic manipulation of PRRSV; FMDVP12A3C gene of serotype O was synthesized and used for an antigen. MARC-145 cells (African green monkey kidney epithelial cell line) were used for electroporation mediated transfection. The transfection or the expression of P12A3C and N protein of PRRSV was analyzed by either replicon containing PRRSV alone or by co-infection of helper PRRSV. Results We constructed PRRSVK418DM replicon vector containing FMDVP12A3C, and genome sequences were confirmed by subsequent sequence analysis. In vitro expression of P12A3C and PRRSV N protein was confirmed by immunofluorescence antibody assay using antibodies specific for PRRSV N protein (anti-PRRSV N MAb), FMDV-VP1 (anti-VP1 MAb). Conclusion The results indicate that PRRSV replicon vector can be a promising novel vector system to control FMDV and useful for vaccine development in the future. PMID:24427767

  17. Characterization of Multidrug-Resistant Escherichia coli by Plasmid Replicon Typing and Pulsed-Field Gel Electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Characterization of plasmids has particular clinical significance because genes encoding important traits such as antimicrobial resistance are frequently present in plasmids. Plasmid replicon typing is a multiplex PCR based method that can be used to classify 18 of the 26 known plasmid t...

  18. Expression of the Novel Hepatitis C Virus Core+1/ARF Protein in the Context of JFH1-Based Replicons

    PubMed Central

    Kotta-Loizou, Ioly; Karakasiliotis, Ioannis; Vassilaki, Niki; Sakellariou, Panagiotis; Bartenschlager, Ralf

    2015-01-01

    Hepatitis C virus contains a second open reading frame within the core gene, designated core+1/ARF. Here we demonstrate for the first time expression of core+1/ARF protein in the context of a bicistronic JFH1-based replicon and report the production of two isoforms, core+1/L (long) and core+1/S (short), with different kinetics. PMID:25694591

  19. Environmental surveillance of poliovirus and non-polio enterovirus in urban sewage in Dakar, Senegal (2007-2013)

    PubMed Central

    Ndiaye, Abdou Kader; Diop, Pape Amadou Mbathio; Diop, Ousmane Madiagne

    2014-01-01

    Introduction Global poliomyelitis eradication initiative relies on (i) laboratory based surveillance of acute flaccid surveillance (AFP) to monitor the circulation of wild poliovirus in a population, and (ii) vaccination to prevent its diffusion. However, as poliovirus can survive in the environment namely in sewage, environmental surveillance (ES) is of growing importance as the eradication target is close. This study aimed to assess polioviruses and non polio enteroviruses circulation in sewage drains covering a significant population of Dakar. Methods From April 2007 to May 2013, 271 specimens of raw sewage were collected using the grab method in 6 neighborhoods of Dakar. Samples were processed to extract and concentrate viruses using polyethylene glycol and Dextran (two-phase separation method). Isolation of enteroviruses was attempted in RD, L20B and Hep2 cell lines. Polioviruses were identified by RT-PCR and Elisa. Non Polio Enteroviruses (NPEVs) were identified by RT-PCR and microneutralisation tests. Results Polioviruses and NPEVs were respectively detected in 34,3% and 42,8% sewage samples. No wild poliovirus neither circulating vaccine-derived Poliovirus (cVDPV) was detected. Neutralization assays have identified 49 non polio enteroviruses that were subsequently classified in 13 serotypes belonging to HEV-A (22, 4%), HEV-B (12, 24%), HEV-C (26, 53%) and HEV-D (6, 12%) species. Conclusion This study is the first documentation of enteroviruses environmental detection in Senegal. It shows the usefulness of environmental surveillance for indirect monitoring of the circulation and distribution of enteroviruses in the community. PMID:25848458

  20. A Review of Historical Naked-Eye Sungrazing Comets

    NASA Astrophysics Data System (ADS)

    Knight, Matthew M.

    2013-10-01

    With the upcoming perihelion passage of Comet ISON (C/2012 S1) in November 2013, there is considerable interest in sungrazing comets at present. However, given the infrequency with which such comets appear, there have been few systematic studies of their behavior near perihelion. Bright, sometimes even daytime-observable comets have been recorded by observers around the world for more than 2000 years. A number of authors have compiled records of possible historical sungrazing comets, e.g., Hasegawa 1979, Kronk 1999, Hasegawa & Nakano 2001, England 2002, and Strom 2002. We review this literature to estimate the frequency of arrival of naked-eye sungrazing comets and investigate if there are any trends in their behavior, such as between peak brightness and length of time a comet is observable. We will also review the “modern” observations of naked-eye sungrazing comets (primarily Kreutz group comets observed since 1800) to investigate the frequency of fragmentation near the Sun and the dependence of survivability on size (as inferred from brightness) and/or perihelion distance.

  1. Effects of activated aflatoxin B/sub 1/ and caffeine on DNA replicon initiation in HeLa cells

    SciTech Connect

    Cramer, P.; Painter, R.B.

    1981-01-01

    Afatoxin B/sub 1/ (AFB/sub 1/) is activated by a rat microsomal extract (S-9) to form a product that inhibits DNA synthesis in HeLa cells. At 10/sup -7/ M, AFB/sub 1/ inhibited initiation of replicons, as shown in alkaline sucrose gradient profiles 30 min after incubation with the drug. Ninety minutes later, the profile of treated cells was similar to that of control, but 4 h later there was another effect on replicon initiation. At 10/sup -6/ M, the inhibition of initiation was greater than at 10/sup -7/ M and increased progressively. Four hours after removal of the drug, the gradient profile showed low amounts of radioactivity in all size classes of DNA. When cells were incubated in medium containing caffeine (2 mM) even as late as 60 min after incubation with AFB/sub 1/, the inhibition of replicon initiation was prevented. If caffeine was later removed from the medium, replicon initiation was then inhibited. At 10/sup -7/ M or 10/sup -6/ M, AFB/sub 1/ had little immediate effect on chain elongation, but at 10/sup -5/ M, the gradient profiles showed an accumulation of low molecular weight DNA molecules, with no radioactivity in the region of high molecular weight DNA, owing to a block to chain elongation; this was not affected by caffeine. These results suggest that AFB/sub 1/ induces damage that changes the fonformation of chromatin so that initiation of new replicons cannot occur; in the presence of caffeine this change does not occur and DNA replication is not inhibited.

  2. Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2

    PubMed Central

    Utt, Age; Das, Pratyush Kumar; Varjak, Margus; Lulla, Valeria; Lulla, Aleksei

    2014-01-01

    ABSTRACT Chikungunya virus (CHIKV) (genus Alphavirus) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E116K (EK) substitution or a GEEGS sequence insertion after residue T648 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels. IMPORTANCE CHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the

  3. Predictive spatial risk model of poliovirus to aid prioritization and hasten eradication in Nigeria

    PubMed Central

    2014-01-01

    Background One of the challenges facing the Global Polio Eradication Initiative is efficiently directing limited resources, such as specially trained personnel, community outreach activities, and satellite vaccinator tracking, to the most at-risk areas to maximize the impact of interventions. A validated predictive model of wild poliovirus circulation would greatly inform prioritization efforts by accurately forecasting areas at greatest risk, thus enabling the greatest effect of program interventions. Methods Using Nigerian acute flaccid paralysis surveillance data from 2004-2013, we developed a spatial hierarchical Poisson hurdle model fitted within a Bayesian framework to study historical polio caseload patterns and forecast future circulation of type 1 and 3 wild poliovirus within districts in Nigeria. A Bayesian temporal smoothing model was applied to address data sparsity underlying estimates of covariates at the district level. Results We find that calculated vaccine-derived population immunity is significantly negatively associated with the probability and number of wild poliovirus case(s) within a district. Recent case information is significantly positively associated with probability of a case, but not the number of cases. We used lagged indicators and coefficients from the fitted models to forecast reported cases in the subsequent six-month periods. Over the past three years, the average predictive ability is 86 ± 2% and 85 ± 4% for wild poliovirus type 1 and 3, respectively. Interestingly, the predictive accuracy of historical transmission patterns alone is equivalent (86 ± 2% and 84 ± 4% for type 1 and 3, respectively). We calculate uncertainty in risk ranking to inform assessments of changes in rank between time periods. Conclusions The model developed in this study successfully predicts districts at risk for future wild poliovirus cases in Nigeria. The highest predicted district risk was 12.8 WPV1 cases in 2006, while the lowest district risk

  4. High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat.

    PubMed

    Tian, Xiao; Azpurua, Jorge; Hine, Christopher; Vaidya, Amita; Myakishev-Rempel, Max; Ablaeva, Julia; Mao, Zhiyong; Nevo, Eviatar; Gorbunova, Vera; Seluanov, Andrei

    2013-07-18

    The naked mole rat (Heterocephalus glaber) displays exceptional longevity, with a maximum lifespan exceeding 30 years. This is the longest reported lifespan for a rodent species and is especially striking considering the small body mass of the naked mole rat. In comparison, a similarly sized house mouse has a maximum lifespan of 4 years. In addition to their longevity, naked mole rats show an unusual resistance to cancer. Multi-year observations of large naked mole-rat colonies did not detect a single incidence of cancer. Here we identify a mechanism responsible for the naked mole rat's cancer resistance. We found that naked mole-rat fibroblasts secrete extremely high-molecular-mass hyaluronan (HA), which is over five times larger than human or mouse HA. This high-molecular-mass HA accumulates abundantly in naked mole-rat tissues owing to the decreased activity of HA-degrading enzymes and a unique sequence of hyaluronan synthase 2 (HAS2). Furthermore, the naked mole-rat cells are more sensitive to HA signalling, as they have a higher affinity to HA compared with mouse or human cells. Perturbation of the signalling pathways sufficient for malignant transformation of mouse fibroblasts fails to transform naked mole-rat cells. However, once high-molecular-mass HA is removed by either knocking down HAS2 or overexpressing the HA-degrading enzyme, HYAL2, naked mole-rat cells become susceptible to malignant transformation and readily form tumours in mice. We speculate that naked mole rats have evolved a higher concentration of HA in the skin to provide skin elasticity needed for life in underground tunnels. This trait may have then been co-opted to provide cancer resistance and longevity to this species. PMID:23783513

  5. 5' and 3' Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves.

    PubMed

    Diamos, Andrew G; Rosenthal, Sun H; Mason, Hugh S

    2016-01-01

    We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5' untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5' UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5' UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody

  6. Antibiotic resistance due to an unusual ColE1-type replicon plasmid in Aeromonas salmonicida.

    PubMed

    Vincent, Antony T; Emond-Rheault, Jean-Guillaume; Barbeau, Xavier; Attéré, Sabrina A; Frenette, Michel; Lagüe, Patrick; Charette, Steve J

    2016-06-01

    Aeromonas salmonicida subsp. salmonicida is a fish pathogen known to have a rich plasmidome. In the present study, we discovered an isolate of this bacterium bearing an additional unidentified small plasmid. After having sequenced the DNA of that isolate by next-generation sequencing, it appeared that the new small plasmid is a ColE1-type replicon plasmid, named here pAsa7. This plasmid bears a functional chloramphenicol-acetyltransferase-encoding gene (cat-pAsa7) previously unknown in A. salmonicida and responsible for resistance to chloramphenicol. A comparison of pAsa7 with pAsa2, the only known ColE1-type replicon plasmid usually found in A. salmonicida subsp. salmonicida, revealed that even if both plasmids share a high structural similarity, it is still unclear if pAsa7 is a derivative of pAsa2 since they showed several mutations at the nucleotide level. Transcriptomic analysis revealed that the cat-pAsa4 gene, another chloramphenicol-acetyltransferase-encoding gene, found on the large plasmid pAsa4, was significantly more transcribed than cat-pAsa7. This was correlated with a higher chloramphenicol resistance for isolates bearing pAsa4 compared with the one having pAsa7. Finally, a phylogenetic analysis showed that both CAT-pAsa4 and CAT-pAsa7 proteins were in different clusters. The clustering was supported by the identity of residues involved in the catalytic site. In addition, to give a better understanding of the large drug-resistance panel of A. salmonicida, this study reinforces the hypothesis that A. salmonicida subsp. salmonicida is a considerable reservoir for mobile genetic elements such as plasmids. PMID:27028891

  7. Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro.

    PubMed Central

    Flanegan, J B; Van Dyke, T A

    1979-01-01

    A soluble RNA-dependent RNA polymerase was isolated from poliovirus-infected HeLa cells and was shown to copy poliovirus RNA in vitro. The enzyme was purified from a 200,000-X-g supernatant of a cytoplasmic extract of infected cells. The activity of the enzyme was measured throughout the purification by using a polyadenylic acid template and oligouridylic acid primer. The enzyme was partially purified by ammonium sulfate precipitation, glycerol gradient centrifugation, and phosphocellulose chromatography. The polymerase precipitated in a 35% saturated solution of ammonium sulfate, sedimented at about 7S on a glycerol gradient, and eluted from phosphocellulose with 0.15 M KC1. The polymerase was purified about 40-fold and was shown to be totally dependent on exogenous RNA for activity and relatively free of contaminating nuclease. The partially purified polymerase was able to use purified polio virion RNA as well as a template. Under the reaction conditions used, the polymerase required an oligouridylic acid primer and all four ribonucleside triphosphates for activity. The optimum ratio of oligouridylic acid molecules to poliovirus RNA molecules for priming activity was about 16:1. A nearest-neighbor analysis of the in vitro RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA rendered it resistant to RNase digestion, thus suggesting that the product RNA was complementary to the virion RNA template. PMID:232168

  8. Attenuation of Neurovirulence, Biodistribution, and Shedding of a Poliovirus:Rhinovirus Chimera after Intrathalamic Inoculation in Macaca fascicularis

    PubMed Central

    Dobrikova, Elena Y.; Goetz, Christian; Walters, Robert W.; Lawson, Sarah K.; Peggins, James O.; Muszynski, Karen; Ruppel, Sheryl; Poole, Karyol; Giardina, Steven L.; Vela, Eric M.; Estep, James E.

    2012-01-01

    A dependence of poliovirus on an unorthodox translation initiation mode can be targeted selectively to drive viral protein synthesis and cytotoxicity in malignant cells. Transformed cells are naturally susceptible to poliovirus, due to widespread ectopic upregulation of the poliovirus receptor, Necl-5, in ectodermal/neuroectodermal cancers. Viral tumor cell killing and the host immunologic response it engenders produce potent, lasting antineoplastic effects in animal tumor models. Clinical application of this principle depends on unequivocal demonstration of safety in primate models for paralytic poliomyelitis. We conducted extensive dose-range-finding, toxicity, biodistribution, shedding, and neutralizing antibody studies of the prototype oncolytic poliovirus recombinant, PVS-RIPO, after intrathalamic inoculation in Macaca fascicularis. These studies suggest that intracerebral PVS-RIPO inoculation does not lead to viral propagation in the central nervous system (CNS), does not cause histopathological CNS lesions or neurological symptoms that can be attributed to the virus, is not associated with extraneural virus dissemination or replication and does not induce shedding of virus with stool. Intrathalamic PVS-RIPO inoculation induced neutralizing antibody responses against poliovirus serotype 1 in all animals studied. PMID:22171271

  9. A poliovirus hybrid expressing a neutralization epitope from the major outer membrane protein of Chlamydia trachomatis is highly immunogenic.

    PubMed Central

    Murdin, A D; Su, H; Manning, D S; Klein, M H; Parnell, M J; Caldwell, H D

    1993-01-01

    Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes on the major outer membrane protein (MOMP) of C. trachomatis have been identified as important targets for the development of vaccines. In order to examine the immunogenicity of a recombinant vector expressing a chlamydial epitope, a poliovirus hybrid was constructed in which part of neutralization antigenic site I of poliovirus type 1 Mahoney (PV1-M) was replaced by a sequence from variable domain I of the MOMP of C. trachomatis serovar A. The chlamydial sequence included the neutralization epitope VAGLEK. This hybrid was viable, grew very well compared with PV1-M, and expressed both poliovirus and chlamydial antigenic determinants. When inoculated into rabbits, this hybrid was highly immunogenic, inducing a strong response against both PV1-M and C. trachomatis serovar A. Antichlamydia titers were 10- to 100-fold higher than the titers induced by equimolar amounts of either purified MOMP or a synthetic peptide expressing the VAGLEK epitope. Furthermore, rabbit antisera raised against this hybrid neutralized chlamydial infectivity both in vitro, for hamster kidney cells, and passively in vivo, for conjunctival epithelia of cynomolgus monkeys. Because poliovirus infection induces a strong mucosal immune response in primates and humans, these results indicate that poliovirus-chlamydia hybrids could become powerful tools for the study of mucosal immunity to chlamydial infection and for the development of recombinant chlamydial vaccines. Images PMID:7691749

  10. Stool screening of Syrian refugees and asylum seekers in Germany, 2013/2014: Identification of Sabin like polioviruses.

    PubMed

    Böttcher, Sindy; Neubauer, Katrin; Baillot, Armin; Rieder, Gabriele; Adam, Maja; Diedrich, Sabine

    2015-10-01

    Germany is a partner of the Global Polio Eradication Initiative. Assurance of polio free status is based on enterovirus surveillance, which focuses on patients with signs of acute flaccid paralysis or aseptic meningitis/encephalitis, representing the key symptoms of poliovirus infection. In response to the wild poliovirus outbreak in Syria 2013 and high number of refugees coming from Syria to Germany, stool samples from 629 Syrian refugees/asylum seekers aged <3 years were screened for wild poliovirus between November 2013 and April 2014. Ninety-three samples (14.8%) were positive in an enterovirus specific PCR. Of these, 12 contained Sabin-like polioviruses. The remaining 81 samples were characterized as non-polio enteroviruses representing several members of groups A-C as well as rhinovirus. Wild-type poliovirus was not detected via stool screening involving molecular and virological methods, indicating a very low risk for the importation by Syrian refugees and asylum seekers at that time. PMID:26321005

  11. Naked singularities in non-self-similar gravitational collapse of radiation shells

    SciTech Connect

    Joshi, P.S.; Dwivedi, I.H. )

    1992-03-15

    Non-self-similar gravitational collapse of imploding radiation is shown to give rise to a strong curvature naked singularity. The conditions are specified for the singularity to be globally naked and the strength of the same is examined along nonspacelike curves and along all the families of nonspacelike geodesics terminating at the singularity in the past.

  12. Trial watch: Naked and vectored DNA-based anticancer vaccines

    PubMed Central

    Bloy, Norma; Buqué, Aitziber; Aranda, Fernando; Castoldi, Francesca; Eggermont, Alexander; Cremer, Isabelle; Sautès-Fridman, Catherine; Fucikova, Jitka; Galon, Jérôme; Spisek, Radek; Tartour, Eric; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2015-01-01

    One type of anticancer vaccine relies on the administration of DNA constructs encoding one or multiple tumor-associated antigens (TAAs). The ultimate objective of these preparations, which can be naked or vectored by non-pathogenic viruses, bacteria or yeast cells, is to drive the synthesis of TAAs in the context of an immunostimulatory milieu, resulting in the (re-)elicitation of a tumor-targeting immune response. In spite of encouraging preclinical results, the clinical efficacy of DNA-based vaccines employed as standalone immunotherapeutic interventions in cancer patients appears to be limited. Thus, efforts are currently being devoted to the development of combinatorial regimens that allow DNA-based anticancer vaccines to elicit clinically relevant immune responses. Here, we discuss recent advances in the preclinical and clinical development of this therapeutic paradigm. PMID:26155408

  13. Sporadic Isolation of Sabin-Like Polioviruses and High-Level Detection of Non-Polio Enteroviruses during Sewage Surveillance in Seven Italian Cities, after Several Years of Inactivated Poliovirus Vaccination

    PubMed Central

    Battistone, A.; Buttinelli, G.; Fiore, S.; Amato, C.; Bonomo, P.; Patti, A. M.; Vulcano, A.; Barbi, M.; Binda, S.; Pellegrinelli, L.; Tanzi, M. L.; Affanni, P.; Castiglia, P.; Germinario, C.; Mercurio, P.; Cicala, A.; Triassi, M.; Pennino, F.

    2014-01-01

    Sewage surveillance in seven Italian cities between 2005 and 2008, after the introduction of inactivated poliovirus vaccination (IPV) in 2002, showed rare polioviruses, none that were wild-type or circulating vaccine-derived poliovirus (cVDPV), and many other enteroviruses among 1,392 samples analyzed. Two of five polioviruses (PV) detected were Sabin-like PV2 and three PV3, based on enzyme-linked immunosorbent assay (ELISA) and PCR results. Neurovirulence-related mutations were found in the 5′ noncoding region (5′NCR) of all strains and, for a PV2, also in VP1 region 143 (Ile > Thr). Intertypic recombination in the 3D region was detected in a second PV2 (Sabin 2/Sabin 1) and a PV3 (Sabin 3/Sabin 2). The low mutation rate in VP1 for all PVs suggests limited interhuman virus passages, consistent with efficient polio immunization in Italy. Nonetheless, these findings highlight the risk of wild or Sabin poliovirus reintroduction from abroad. Non-polio enteroviruses (NPEVs) were detected, 448 of which were coxsackievirus B (CVB) and 294 of which were echoviruses (Echo). Fifty-six NPEVs failing serological typing were characterized by sequencing the VP1 region (nucleotides [nt] 2628 to 2976). A total of 448 CVB and 294 Echo strains were identified; among those strains, CVB2, CVB5, and Echo 11 predominated. Environmental CVB5 and CVB2 strains from this study showed high sequence identity with GenBank global strains. The high similarity between environmental NPEVs and clinical strains from the same areas of Italy and the same periods indicates that environmental strains reflect the viruses circulating in the population and highlights the potential risk of inefficient wastewater treatments. This study confirmed that sewage surveillance can be more sensitive than acute flaccid paralysis (AFP) surveillance in monitoring silent poliovirus circulation in the population as well as the suitability of molecular approaches to enterovirus typing. PMID:24814793

  14. Sporadic isolation of sabin-like polioviruses and high-level detection of non-polio enteroviruses during sewage surveillance in seven Italian cities, after several years of inactivated poliovirus vaccination.

    PubMed

    Battistone, A; Buttinelli, G; Fiore, S; Amato, C; Bonomo, P; Patti, A M; Vulcano, A; Barbi, M; Binda, S; Pellegrinelli, L; Tanzi, M L; Affanni, P; Castiglia, P; Germinario, C; Mercurio, P; Cicala, A; Triassi, M; Pennino, F; Fiore, L

    2014-08-01

    Sewage surveillance in seven Italian cities between 2005 and 2008, after the introduction of inactivated poliovirus vaccination (IPV) in 2002, showed rare polioviruses, none that were wild-type or circulating vaccine-derived poliovirus (cVDPV), and many other enteroviruses among 1,392 samples analyzed. Two of five polioviruses (PV) detected were Sabin-like PV2 and three PV3, based on enzyme-linked immunosorbent assay (ELISA) and PCR results. Neurovirulence-related mutations were found in the 5'noncoding region (5'NCR) of all strains and, for a PV2, also in VP1 region 143 (Ile>Thr). Intertypic recombination in the 3D region was detected in a second PV2 (Sabin 2/Sabin 1) and a PV3 (Sabin 3/Sabin 2). The low mutation rate in VP1 for all PVs suggests limited interhuman virus passages, consistent with efficient polio immunization in Italy. Nonetheless, these findings highlight the risk of wild or Sabin poliovirus reintroduction from abroad. Non-polio enteroviruses (NPEVs) were detected, 448 of which were coxsackievirus B (CVB) and 294 of which were echoviruses (Echo). Fifty-six NPEVs failing serological typing were characterized by sequencing the VP1 region (nucleotides [nt] 2628 to 2976). A total of 448 CVB and 294 Echo strains were identified; among those strains, CVB2, CVB5, and Echo 11 predominated. Environmental CVB5 and CVB2 strains from this study showed high sequence identity with GenBank global strains. The high similarity between environmental NPEVs and clinical strains from the same areas of Italy and the same periods indicates that environmental strains reflect the viruses circulating in the population and highlights the potential risk of inefficient wastewater treatments. This study confirmed that sewage surveillance can be more sensitive than acute flaccid paralysis (AFP) surveillance in monitoring silent poliovirus circulation in the population as well as the suitability of molecular approaches to enterovirus typing. PMID:24814793

  15. Multiple Independent Emergences of Type 2 Vaccine-Derived Polioviruses during a Large Outbreak in Northern Nigeria

    PubMed Central

    Shaw, Jing; Jorba, Jaume; Bukbuk, David; Adu, Festus; Gumede, Nicksy; Pate, Muhammed Ali; Abanida, Emmanuel Ade; Gasasira, Alex; Iber, Jane; Chen, Qi; Vincent, Annelet; Chenoweth, Paul; Henderson, Elizabeth; Wannemuehler, Kathleen; Naeem, Asif; Umami, Rifqiyah Nur; Nishimura, Yorihiro; Shimizu, Hiroyuki; Baba, Marycelin; Adeniji, Adekunle; Williams, A. J.; Kilpatrick, David R.; Oberste, M. Steven; Wassilak, Steven G.; Tomori, Oyewale; Pallansch, Mark A.; Kew, Olen

    2013-01-01

    Since 2005, a large poliomyelitis outbreak associated with type 2 circulating vaccine-derived poliovirus (cVDPV2) has occurred in northern Nigeria, where immunization coverage with trivalent oral poliovirus vaccine (tOPV) has been low. Phylogenetic analysis of P1/capsid region sequences of isolates from each of the 403 cases reported in 2005 to 2011 resolved the outbreak into 23 independent type 2 vaccine-derived poliovirus (VDPV2) emergences, at least 7 of which established circulating lineage groups. Virus from one emergence (lineage group 2005-8; 361 isolates) was estimated to have circulated for over 6 years. The population of the major cVDPV2 lineage group expanded rapidly in early 2009, fell sharply after two tOPV rounds in mid-2009, and gradually expanded again through 2011. The two major determinants of attenuation of the Sabin 2 oral poliovirus vaccine strain (A481 in the 5′-untranslated region [5′-UTR] and VP1-Ile143) had been replaced in all VDPV2 isolates; most A481 5′-UTR replacements occurred by recombination with other enteroviruses. cVDPV2 isolates representing different lineage groups had biological properties indistinguishable from those of wild polioviruses, including efficient growth in neuron-derived HEK293 cells, the capacity to cause paralytic disease in both humans and PVR-Tg21 transgenic mice, loss of the temperature-sensitive phenotype, and the capacity for sustained person-to-person transmission. We estimate from the poliomyelitis case count and the paralytic case-to-infection ratio for type 2 wild poliovirus infections that ∼700,000 cVDPV2 infections have occurred during the outbreak. The detection of multiple concurrent cVDPV2 outbreaks in northern Nigeria highlights the risks of cVDPV emergence accompanying tOPV use at low rates of coverage in developing countries. PMID:23408630

  16. Twenty-Eight Years of Poliovirus Replication in an Immunodeficient Individual: Impact on the Global Polio Eradication Initiative

    PubMed Central

    Dunn, Glynis; Klapsa, Dimitra; Wilton, Thomas; Stone, Lindsay; Minor, Philip D.; Martin, Javier

    2015-01-01

    There are currently huge efforts by the World Health Organization and partners to complete global polio eradication. With the significant decline in poliomyelitis cases due to wild poliovirus in recent years, rare cases related to the use of live-attenuated oral polio vaccine assume greater importance. Poliovirus strains in the oral vaccine are known to quickly revert to neurovirulent phenotype following replication in humans after immunisation. These strains can transmit from person to person leading to poliomyelitis outbreaks and can replicate for long periods of time in immunodeficient individuals leading to paralysis or chronic infection, with currently no effective treatment to stop excretion from these patients. Here, we describe an individual who has been excreting type 2 vaccine-derived poliovirus for twenty eight years as estimated by the molecular clock established with VP1 capsid gene nucleotide sequences of serial isolates. This represents by far the longest period of excretion described from such a patient who is the only identified individual known to be excreting highly evolved vaccine-derived poliovirus at present. Using a range of in vivo and in vitro assays we show that the viruses are very virulent, antigenically drifted and excreted at high titre suggesting that such chronic excreters pose an obvious risk to the eradication programme. Our results in virus neutralization assays with human sera and immunisation-challenge experiments using transgenic mice expressing the human poliovirus receptor indicate that while maintaining high immunisation coverage will likely confer protection against paralytic disease caused by these viruses, significant changes in immunisation strategies might be required to effectively stop their occurrence and potential widespread transmission. Eventually, new stable live-attenuated polio vaccines with no risk of reversion might be required to respond to any poliovirus isolation in the post-eradication era. PMID:26313548

  17. Conditional poliovirus mutants made by random deletion mutagenesis of infectious cDNA.

    PubMed Central

    Kirkegaard, K; Nelsen, B

    1990-01-01

    Small deletions were introduced into DNA plasmids bearing cDNA copies of Mahoney type 1 poliovirus RNA. The procedure used was similar to that of P. Hearing and T. Shenk (J. Mol. Biol. 167:809-822, 1983), with modifications designed to introduce only one lesion randomly into each DNA molecule. Methods to map small deletions in either large DNA or RNA molecules were employed. Two poliovirus mutants, VP1-101 and VP1-102, were selected from mutagenized populations on the basis of their host range phenotype, showing a large reduction in the relative numbers of plaques on CV1 and HeLa cells compared with wild-type virus. The deletions borne by the mutant genomes were mapped to the region encoding the amino terminus of VP1. That these lesions were responsible for the mutant phenotypes was substantiated by reintroduction of the sequenced lesions into a wild-type poliovirus cDNA by deoxyoligonucleotide-directed mutagenesis. The deletion of nucleotides encoding amino acids 8 and 9 of VP1 was responsible for the VP1-101 phenotype; the VP1-102 defect was caused by the deletion of the sequences encoding the first four amino acids of VP1. The peptide sequence at the VP1-VP3 proteolytic cleavage site was altered from glutamine-glycine to glutamine-methionine in VP1-102; this apparently did not alter the proteolytic cleavage pattern. The biochemical defects resulting from these mutations are discussed in the accompanying report. Images PMID:2152811

  18. The human poliovirus receptor. Receptor-virus interaction and parameters of disease specificity.

    PubMed

    Gromeier, M; Lu, H H; Bernhardt, G; Harber, J J; Bibb, J A; Wimmer, E

    1995-05-25

    The host range of poliovirus is determined by the expression of the hPVR, a member of the immunoglobulin superfamily. We characterized hPVR proteins biochemically and found them to be complex-type glycoproteins. The outermost V-like domain of three extracellular domains harbors the PVR function. A panel of single or multiple amino acid exchanges were introduced throughout this domain in order to localize regions involved in virus-receptor interactions. Putative contact amino acids were found to reside in the C'C"D and DE regions. Binding and uptake of poliovirus paralleled virus replication in all mutants tested suggesting that virus binding was affected without abrogating the ability to mediate subsequent events in the infection. Although the primate PVR is essential in conferring susceptibility to poliovirus infection, certain strains can induce neurological disease in rodents. Mouse neurovirulent PV isolates of divergent serotypical origin each provoked a distinctive, characteristic neurological syndrome upon intracerebral infection of wild-type mice. We analyzed clinical and histopathological features of diffuse encephalomyelitis caused by these PV strains and compared the condition with poliomyelitis in mice transgenic for the hPVR. Diffuse PV encephalomyelitis in wild-type mice could be distinguished clinically and histopathologically from hPVR-mediated poliomyelitis in trangenic mice. We localized the determinants of mouse neurovirulence of PV1(LS-a), a derivative of PV1 (Mahoney), in a portion of the viral genome encompassing parts of the capsid protein VP1 as well as the nonstructural protein 2A. Mouse neuropathogenicity could possibly be conferred by reduced particle stability of PV1(LS-a) inasmuch as we found particles to be thermolabile. PMID:7611627

  19. Serial Recombination during Circulation of Type 1 Wild-Vaccine Recombinant Polioviruses in China

    PubMed Central

    Liu, Hong-Mei; Zheng, Du-Ping; Zhang, Li-Bi; Oberste, M. Steven; Kew, Olen M.; Pallansch, Mark A.

    2003-01-01

    Type 1 wild-vaccine recombinant polioviruses sharing a 367-nucleotide (nt) block of Sabin 1-derived sequence spanning the VP1 and 2A genes circulated widely in China from 1991 to 1993. We surveyed the sequence relationships among 34 wild-vaccine recombinants by comparing six genomic intervals: the conserved 5′-untranslated region (5′-UTR) (nt 186 to 639), the hypervariable portion of the 5′-UTR (nt 640 to 742), the VP4 and partial VP2 genes (nt 743 to 1176), the VP1 gene (nt 2480 to 3385), the 2A gene (nt 3386 to 3832), and the partial 3D gene (nt 6011 to 6544). The 5′-UTR, capsid (VP4-VP2 and VP1), and 2A sequence intervals had similar phylogenies. By contrast, the partial 3D sequences could be distributed into five divergent genetic classes. Most (25 of 34) of the wild-vaccine recombinant isolates showed no evidence of additional recombination beyond the initial wild-Sabin recombination event. Eight isolates from 1992 to 1993, however, appear to be derived from three independent additional recombination events, and one 1993 isolate was derived from two consecutive events. Complete genomic sequences of a representative isolate for each 3D sequence class demonstrated that these exchanges had occurred in the 2B, 2C, and 3D genes. The 3D gene sequences were not closely related to those of the Sabin strains or 53 diverse contemporary wild poliovirus isolates from China, but all were related to the 3D genes of species C enteroviruses. The appearance within approximately 2.5 years of five recombinant classes derived from a single ancestral infection illustrates the rapid emergence of new recombinants among circulating wild polioviruses. PMID:14512548

  20. Efficiency of beef extract for the recovery of poliovirus from wastewater effluents.

    PubMed

    Landry, E F; Vaughn, J M; Thomas, M Z; Vicale, T J

    1978-10-01

    The efficiency of poliovirus elution from fiber glass cartridge filters (K27), epoxy-fiber glass-asbestos filters (M780), and pleated cartridge filters was assessed by using 3% beef extract (pH 9.0) or 0.1 M glycine (pH 11.5). Poliovirus type I, strain LSc, was seeded into 20- to 25-gallon (ca. 75.6- to 95.6-liter) samples of treated sewage effluent and concentrated by using a filter adsorption-elution technique. Virus elution was accomplished by using either two 600-ml portions of 3% beef extract (pH 9.0), or two 1-liter portions of 0.1 M glycine (pH 11.5). In all experiments, beef extract elution followed by organic flocculation was found to be superior, yielding a mean recovery efficiency of 85%, with recoveries ranging from 68 to 100%. Elution with 0.1 M glycine (pH 11.5) followed by inorganic flocculation resulted in a mean recovery efficiency of 36%. The variable range of recoveries with beef extract could not be significantly improved by varying the type of beef extract or by extending the elution time to 30 min. Second-step reconcentration of 1-liter seeded sewage effluent and renovated wastewater samples indicated that organic flocculation was a more efficient method for virus recovery than inorganic flocculation. Beef extract concentrations of less than 3% were found to be efficient in the recovery of poliovirus from renovated wastewater. PMID:30391

  1. In Vitro Synthesis of Poliovirus Ribonucleic Acid: Role of the Replicative Intermediate

    PubMed Central

    Girard, Marc

    1969-01-01

    Poliovirus ribonucleic acid (RNA) polymerase crude extracts could be stored frozen in liquid nitrogen without loss of activity or specificity. The major in vitro product of these extracts was viral single-stranded RNA. However, after short periods of incubation with radioactive nucleoside triphosphates, most of the incorporated label was found in replicative intermediate. When excess unlabeled nucleoside triphosphate was added, the label was displaced from the replicative intermediate and accumulated as viral RNA. It is concluded from this experiment that the replicative intermediate is the precursor to viral RNA. In addition, some of the label was chased into double-stranded RNA. The implications of this finding are discussed. PMID:4306193

  2. Calcium Flux between the Endoplasmic Reticulum and Mitochondrion Contributes to Poliovirus-Induced Apoptosis▿

    PubMed Central

    Brisac, Cynthia; Téoulé, François; Autret, Arnaud; Pelletier, Isabelle; Colbère-Garapin, Florence; Brenner, Catherine; Lemaire, Christophe; Blondel, Bruno

    2010-01-01

    We show that poliovirus (PV) infection induces an increase in cytosolic calcium (Ca2+) concentration in neuroblastoma IMR5 cells, at least partly through Ca2+ release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) channels. This leads to Ca2+ accumulation in mitochondria through the mitochondrial Ca2+ uniporter and the voltage-dependent anion channel (VDAC). This increase in mitochondrial Ca2+ concentration in PV-infected cells leads to mitochondrial dysfunction and apoptosis. PMID:20861253

  3. [Investigation of a Patient with Pre-vaccine-derived Poliovirus in Shandong Province, China].

    PubMed

    Lin, Xiaojuan; Liu, Yao; Wang, Suting; Zhang Xiao; Song, Lizhi; Tao, Zexin; Ji, Feng; Xiong, Ping; Xu, Aiqiang

    2015-09-01

    To analyze the genetic characteristics of a polio-I highly variant vaccine recombinant virus in Shandong Province (China) in 2011 and to identify isolates from healthy contacts, two stool specimens from one patient with acute flaccid paralysis (AFP) and 40 stool specimens from his contacts were collected for virus isolation. The complete genome of poliovirus and VP1 coding region of the non-polio enterovirus were sequenced. Homologous comparison and phylogenetic analyses based on VP1 sequences were undertaken among coxsackievirus (CV) B1, CV-B3 isolates, and those in GenBank. One poliovirus (P1/11186), CV-A4 and CV-A8 were isolated from the AFP patient; one CV-A2, Echovirus 3 (E-3), E-12 and E-14, ten CV-B1, and five CV-B3 strains were isolated from his contacts. These results led us to believe that there may be a human enterovirus epidemic in this area, and that surveillance must be enhanced. P1/11186 was a type-1 vaccine-related poliovirus; it combined with type-2 and type-3 polioviruses in 2A and 3A regions, respectively. There were 25 nucleotide mutations with 9 amino-acid alterations in the entire genome. There were 8 nucleotide mutations with 5 amino-acid alterations in the VP1 region compared with the corresponding Sabin strains. Homology analyses suggested that the ten CV-B1 isolates had 97.0%-100% nucleotide and 98.9%-100% amino-acid identities with each other, as well as 92.6%-100% nucleotide and 99.2%-100% amino-acid identities among the five CV-B3 isolates. Phylogenetic analyses on the complete sequences of VP1 among CV-B1 and CV-B3 isolates showed that Shandong strains, together with strains from other provinces in China, had a close relationship and belonged to the same group. PMID:26738293

  4. Update on Vaccine-Derived Polioviruses - Worldwide, January 2014-March 2015.

    PubMed

    Diop, Ousmane M; Burns, Cara C; Sutter, Roland W; Wassilak, Steven G; Kew, Olen M

    2015-06-19

    Since the World Health Assembly's 1988 resolution to eradicate poliomyelitis, one of the main tools of the World Health Organization (WHO) Global Polio Eradication Initiative (GPEI) has been the live, attenuated oral poliovirus vaccine (OPV). OPV might require several doses to induce immunity but provides long-term protection against paralytic disease. Through effective use of OPV, GPEI has brought polio to the threshold of eradication. Wild poliovirus type 2 (WPV2) was eliminated in 1999, WPV3 has not been detected since November 2012, and WPV1 circulation appears to be restricted to parts of Pakistan and Afghanistan. However, continued use of OPV carries two key risks. The first, vaccine-associated paralytic poliomyelitis (VAPP) has been recognized since the early 1960s. VAPP is a very rare event that occurs sporadically when an administered dose of OPV reverts to neurovirulence and causes paralysis in the vaccine recipient or a nonimmune contact. VAPP can occur among immunologically normal vaccine recipients and their contacts as well as among persons who have primary immunodeficiencies (PIDs) manifested by defects in antibody production; it is not associated with outbreaks. The second, the emergence of genetically divergent, neurovirulent vaccine-derived polioviruses (VDPVs) was recognized more recently. Circulating VDPVs (cVDPVs) resemble WPVs and, in areas with low OPV coverage, can cause polio outbreaks. Immunodeficiency-associated VDPVs (iVDPVs) can replicate and be excreted for years in some persons with PIDs; GPEI maintains a registry of iVDPV cases. Ambiguous VDPVs (aVDPVs) are isolates that cannot be classified definitively. This report updates previous surveillance summaries and describes VDPVs detected worldwide during January 2014-March 2015. Those include new cVDPV outbreaks in Madagascar and South Sudan, and sharply reduced type 2 cVDPV (cVDPV2) circulation in Nigeria and Pakistan during the latter half of 2014. Eight newly identified persons in

  5. A sindbis virus replicon-based DNA vaccine encoding the rabies virus glycoprotein elicits immune responses and complete protection in mice from lethal challenge.

    PubMed

    Saxena, Sonal; Dahiya, Shyam S; Sonwane, Arvind A; Patel, Chhabi Lal; Saini, Mohini; Rai, A; Gupta, Praveen K

    2008-12-01

    A sindbis virus replicon-based DNA vaccine encoding rabies virus glycoprotein (G) was developed by subcloning rabies G gene into a sindbis virus replicon-based vaccine vector (pAlpha). The self-amplification of RNA transcripts and translation efficiency of rabies G was analyzed in pAlpha-Rab-G-transfected mammalian cells using RT-PCR, SDS-PAGE and Western blot analysis. The transfected cells also showed induction of apoptosis which is an important event in the enhancement of immune responses. Further, immune responses induced with replicon-based rabies DNA vaccine (pAlpha-Rab-G) was compared with conventional rabies DNA vaccine and commercial cell culture vaccine (Rabipur) in intramuscularly injected mice. The mice immunized with replicon-based rabies DNA vaccine induced humoral and cell mediated immune responses better than conventional rabies DNA vaccine however, comparable to Rabipur vaccine. On challenge with rabies virus CVS strain, replicon-based rabies DNA vaccine conferred complete protection similar to Rabipur. These results demonstrate that replicon-based rabies DNA vaccine is effective in inducing both humoral and cellular immune responses and can be considered as effective vaccine against rabies. PMID:18848857

  6. Selection and characterization of hepatitis C virus replicons dually resistant to the polymerase and protease inhibitors HCV-796 and boceprevir (SCH 503034).

    PubMed

    Flint, Mike; Mullen, Stanley; Deatly, Anne M; Chen, Wei; Miller, Lynn Z; Ralston, Robert; Broom, Colin; Emini, Emilio A; Howe, Anita Y M

    2009-02-01

    HCV-796 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase, and boceprevir is an inhibitor of the NS3 serine protease. The emergence of replicon variants resistant to the combination of HCV-796 and boceprevir was evaluated. Combining the inhibitors greatly reduced the frequency with which resistant colonies arose; however, some resistant replicon cells could be isolated by the use of low inhibitor concentrations. These replicons were approximately 1,000-fold less susceptible to HCV-796 and 9-fold less susceptible to boceprevir. They also exhibited resistance to anthranilate nonnucleoside inhibitors of NS5B but were fully sensitive to inhibitors of different mechanisms: a pyranoindole, Hsp90 inhibitors, an NS5B nucleoside inhibitor, and pegylated interferon (Peg-IFN). The replicon was cleared from the combination-resistant cells by extended treatment with Peg-IFN. Mutations known to confer resistance to HCV-796 (NS5B C316Y) and boceprevir (NS3 V170A) were present in the combination-resistant replicons. These changes could be selected together and coexist in the same genome. The replicon bearing both changes exhibited reduced sensitivity to inhibition by HCV-796 and boceprevir but had a reduced replicative capacity. PMID:18936191

  7. Selection and Characterization of Hepatitis C Virus Replicons Dually Resistant to the Polymerase and Protease Inhibitors HCV-796 and Boceprevir (SCH 503034) ▿

    PubMed Central

    Flint, Mike; Mullen, Stanley; Deatly, Anne M.; Chen, Wei; Miller, Lynn Z.; Ralston, Robert; Broom, Colin; Emini, Emilio A.; Howe, Anita Y. M.

    2009-01-01

    HCV-796 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase, and boceprevir is an inhibitor of the NS3 serine protease. The emergence of replicon variants resistant to the combination of HCV-796 and boceprevir was evaluated. Combining the inhibitors greatly reduced the frequency with which resistant colonies arose; however, some resistant replicon cells could be isolated by the use of low inhibitor concentrations. These replicons were approximately 1,000-fold less susceptible to HCV-796 and 9-fold less susceptible to boceprevir. They also exhibited resistance to anthranilate nonnucleoside inhibitors of NS5B but were fully sensitive to inhibitors of different mechanisms: a pyranoindole, Hsp90 inhibitors, an NS5B nucleoside inhibitor, and pegylated interferon (Peg-IFN). The replicon was cleared from the combination-resistant cells by extended treatment with Peg-IFN. Mutations known to confer resistance to HCV-796 (NS5B C316Y) and boceprevir (NS3 V170A) were present in the combination-resistant replicons. These changes could be selected together and coexist in the same genome. The replicon bearing both changes exhibited reduced sensitivity to inhibition by HCV-796 and boceprevir but had a reduced replicative capacity. PMID:18936191

  8. Sabin Vaccine Reversion in the Field: a Comprehensive Analysis of Sabin-Like Poliovirus Isolates in Nigeria

    PubMed Central

    Chang, Stewart; Iber, Jane; Zhao, Kun; Adeniji, Johnson A.; Bukbuk, David; Baba, Marycelin; Behrend, Matthew; Burns, Cara C.; Oberste, M. Steven

    2015-01-01

    ABSTRACT To assess the dynamics of genetic reversion of live poliovirus vaccine in humans, we studied molecular evolution in Sabin-like poliovirus isolates from Nigerian acute flaccid paralysis cases obtained from routine surveillance. We employed a novel modeling approach to infer substitution and recombination rates from whole-genome sequences and information about poliovirus infection dynamics and the individual vaccination history. We confirmed observations from a recent vaccine trial that VP1 substitution rates are increased for Sabin-like isolates relative to the rate for the wild type due to increased nonsynonymous substitution rates. We also inferred substitution rates for attenuating nucleotides and confirmed that reversion can occur in days to weeks after vaccination. We combine our observations for Sabin-like virus evolution with the molecular clock for VP1 of circulating wild-type strains to infer that the mean time from the initiating vaccine dose to the earliest detection of circulating vaccine-derived poliovirus (cVDPV) is 300 days for Sabin-like virus type 1, 210 days for Sabin-like virus type 2, and 390 days for Sabin-like virus type 3. Phylogenetic relationships indicated transient local transmission of Sabin-like virus type 3 and, possibly, Sabin-like virus type 1 during periods of low wild polio incidence. Comparison of Sabin-like virus recombinants with known Nigerian vaccine-derived poliovirus recombinants shows that while recombination with non-Sabin enteroviruses is associated with cVDPV, the recombination rates are similar for Sabin isolate-Sabin isolate and Sabin isolate–non-Sabin enterovirus recombination after accounting for the time from dosing to the time of detection. Our study provides a comprehensive picture of the evolutionary dynamics of the oral polio vaccine in the field. IMPORTANCE The global polio eradication effort has completed its 26th year. Despite success in eliminating wild poliovirus from most of the world, polio

  9. Self-Amplifying Replicon RNA Vaccine Delivery to Dendritic Cells by Synthetic Nanoparticles

    PubMed Central

    McCullough, Kenneth C.; Milona, Panagiota; Thomann-Harwood, Lisa; Démoulins, Thomas; Englezou, Pavlos; Suter, Rolf; Ruggli, Nicolas

    2014-01-01

    Dendritic cells (DC) play essential roles determining efficacy of vaccine delivery with respect to immune defence development and regulation. This renders DCs important targets for vaccine delivery, particularly RNA vaccines. While delivery of interfering RNA oligonucleotides to the appropriate intracellular sites for RNA-interference has proven successful, the methodologies are identical for RNA vaccines, which require delivery to RNA translation sites. Delivery of mRNA has benefitted from application of cationic entities; these offer value following endocytosis of RNA, when cationic or amphipathic properties can promote endocytic vesicle membrane perturbation to facilitate cytosolic translocation. The present review presents how such advances are being applied to the delivery of a new form of RNA vaccine, replicons (RepRNA) carrying inserted foreign genes of interest encoding vaccine antigens. Approaches have been developed for delivery to DCs, leading to the translation of the RepRNA and encoded vaccine antigens both in vitro and in vivo. Potential mechanisms favouring efficient delivery leading to translation are discussed with respect to the DC endocytic machinery, showing the importance of cytosolic translocation from acidifying endocytic structures. The review relates the DC endocytic pathways to immune response induction, and the potential advantages for these self-replicating RNA vaccines in the near future. PMID:26344889

  10. Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens

    PubMed Central

    Wilms, Ina; Overlöper, Aaron; Nowrousian, Minou; Sharma, Cynthia M.; Narberhaus, Franz

    2012-01-01

    Agrobacterium species are capable of interkingdom gene transfer between bacteria and plants. The genome of Agrobacterium tumefaciens consists of a circular and a linear chromosome, the At-plasmid and the Ti-plasmid, which harbors bacterial virulence genes required for tumor formation in plants. Little is known about promoter sequences and the small RNA (sRNA) repertoire of this and other α-proteobacteria. We used a differential RNA sequencing (dRNA-seq) approach to map transcriptional start sites of 388 annotated genes and operons. In addition, a total number of 228 sRNAs was revealed from all four Agrobacterium replicons. Twenty-two of these were confirmed by independent RNA gel blot analysis and several sRNAs were differentially expressed in response to growth media, growth phase, temperature or pH. One sRNA from the Ti-plasmid was massively induced under virulence conditions. The presence of 76 cis-antisense sRNAs, two of them on the reverse strand of virulence genes, suggests considerable antisense transcription in Agrobacterium. The information gained from this study provides a valuable reservoir for an in-depth understanding of sRNA-mediated regulation of the complex physiology and infection process of Agrobacterium. PMID:22336765

  11. Remodeling the Endoplasmic Reticulum by Poliovirus Infection and by Individual Viral Proteins: an Autophagy-Like Origin for Virus-Induced Vesicles

    PubMed Central

    Suhy, David A.; Giddings, Thomas H.; Kirkegaard, Karla

    2000-01-01

    All positive-strand RNA viruses of eukaryotes studied assemble RNA replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of Golgi-derived or endosomal membranes in buoyant density gradients, although changes in ionic strength affected ER and virus-induced vesicles, but not other cellular organelles, similarly. When expressed in isolation, two viral proteins of the poliovirus RNA replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B and 2C proteins, membranes identical in buoyant density to those observed during poliovirus infection were formed. When coexpressed with 2BC, viral protein 3A was quantitatively incorporated into these fractions, and the membranes formed were ultrastructurally similar to those in poliovirus-infected cells. These data argue that poliovirus-induced vesicles derive from the ER by the action of viral proteins 2BC and 3A by a mechanism that excludes resident host proteins. The double-membraned morphology, cytosolic content, and apparent ER origin of poliovirus-induced membranes are all consistent with an autophagic origin for these membranes. PMID:10982339

  12. A Critical Role of a Cellular Membrane Traffic Protein in Poliovirus RNA Replication

    PubMed Central

    Belov, George A.; Feng, Qian; Nikovics, Krisztina; Jackson, Catherine L.; Ehrenfeld, Ellie

    2008-01-01

    Replication of many RNA viruses is accompanied by extensive remodeling of intracellular membranes. In poliovirus-infected cells, ER and Golgi stacks disappear, while new clusters of vesicle-like structures form sites for viral RNA synthesis. Virus replication is inhibited by brefeldin A (BFA), implicating some components(s) of the cellular secretory pathway in virus growth. Formation of characteristic vesicles induced by expression of viral proteins was not inhibited by BFA, but they were functionally deficient. GBF1, a guanine nucleotide exchange factor for the small cellular GTPases, Arf, is responsible for the sensitivity of virus infection to BFA, and is required for virus replication. Knockdown of GBF1 expression inhibited virus replication, which was rescued by catalytically active protein with an intact N-terminal sequence. We identified a mutation in GBF1 that allows growth of poliovirus in the presence of BFA. Interaction between GBF1 and viral protein 3A determined the outcome of infection in the presence of BFA. PMID:19023417

  13. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    SciTech Connect

    Andoh, Y.; Yoshii, N.; Yamada, A.; Kojima, H.; Mizutani, K.; Okazaki, S.; Fujimoto, K.; Nakagawa, A.; Nomoto, A.

    2014-10-28

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10{sup 6} all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  14. S2M: A Stochastic Simulation Model of Poliovirus Genetic State Transition

    PubMed Central

    Ecale Zhou, Carol L.

    2016-01-01

    Modeling the molecular mechanisms that govern genetic variation can be useful in understanding the dynamics that drive genetic state transition in quasispecies viruses. For example, there is considerable interest in understanding how the relatively benign vaccine strains of poliovirus eventually revert to forms that confer neurovirulence and cause disease (ie, vaccine-derived poliovirus). This report describes a stochastic simulation model, S2M, which can be used to generate hypothetical outcomes based on known mechanisms of genetic diversity. S2M begins with predefined genotypes based on the Sabin-1 and Mahoney wild-type sequences, constructs a set of independent cell-based populations, and performs in-cell replication and cell-to-cell infection cycles while quantifying genetic changes that track the transition from Sabin-1 toward Mahoney. Realism is incorporated into the model by assigning defaults for variables that constrain mechanisms of genetic variability based roughly on metrics reported in the literature, yet these values can be modified at the command line in order to generate hypothetical outcomes driven by these parameters. To demonstrate the utility of S2M, simulations were performed to examine the effects of the rates of replication error and recombination and the presence or absence of defective interfering particles, upon reaching the end states of Mahoney resemblance (semblance of a vaccine-derived state), neurovirulence, genome fitness, and cloud diversity. Simulations provide insight into how modeled biological features may drive hypothetical outcomes, independently or in combination, in ways that are not always intuitively obvious. PMID:27385911

  15. Effect of Poliovirus on Deoxyribonucleic Acid Synthesis in HeLa Cells

    PubMed Central

    Ackermann, W. W.; Cox, D. C.; Kurtz, H.; Powers, C. D.; Davies, S. J.

    1966-01-01

    Ackermann, W. W. (University of Michigan, Ann Arbor), D. C. Cox, H. Kurtz, C. D. Powers, and S. J. Davies. Effect of poliovirus on deoxyribonucleic acid synthesis in HeLa cells. J. Bacteriol. 91:1943–1952. 1966.—Both poliovirus and arginine stimulated deoxyribonucleic acid (DNA) synthesis in cultures of HeLa cells which were preconditioned by incubation in a medium deficient in arginine. However, the number of cells producing DNA was unaffected. DNA synthesis in such preconditioned cells was 10 to 20% of the maximal value obtained with a full complement of amino acids. Inhibition of DNA synthesis was produced in these cultures either by increasing the multiplicity of exposure above 40 plaque-forming units of virus per cell or by increasing the concentration of the deficient amino acid at the time of virus addition. Inhibition of DNA synthesis resulted from a reduction in the fraction of cells producing DNA. The concentration of arginine required for viral inhibition of DNA synthesis is greater than that for viral multiplication. PMID:4287076

  16. Development of a novel bag-mediated filtration system for environmental recovery of poliovirus.

    PubMed

    Fagnant, Christine Susan; Beck, Nicola Koren; Yang, Ming-Fong; Barnes, Kilala Sayisha; Boyle, David S; Meschke, John Scott

    2014-12-01

    Poliovirus (PV) is on the verge of global eradication. Due to asymptomatic shedding, eradication certification requires environmental and clinical surveillance. Current environmental surveillance methods involve collection and processing of 400-mL to 1-L grab samples by a two-phase separation method, where sample volume limits detection sensitivity. Filtration of larger sample volumes facilitates increased detection sensitivity. This study describes development of a pumpless in-field filtration system for poliovirus recovery from environmental waters. Recovery of PV types 1, 2, and 3 were compared for glass wool, ViroCap, and NanoCeram (PV1 only) filters. Seeded experiments were performed using 10(5) plaque forming units of PV inoculated into 10-L volumes of secondary effluent, surface water, or a 50:50 mixture of each at pH 7.0. Filter eluates were plated onto buffalo green monkey kidney cells for virus enumeration by plaque assay. Across all water types, recovery from glass wool filters for PV1, PV2, and PV3 averaged 17%, 28%, and 6%, respectively. Recovery from ViroCaps for PV1, PV2, and PV3 averaged 44%, 70%, and 81%, respectively. 10-L samples of moderate turbidity water were processed through ViroCap filters in less than 30 minutes using a pumpless, bag-mediated filtration system. Bag-mediated filtration offers a simple, compact, and efficient method for enhanced environmental PV surveillance. PMID:25473984

  17. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    NASA Astrophysics Data System (ADS)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Fujimoto, K.; Kojima, H.; Mizutani, K.; Nakagawa, A.; Nomoto, A.; Okazaki, S.

    2014-10-01

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 106 all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  18. Modification of an adenovirus major late promoter-binding factor during poliovirus infection.

    PubMed Central

    Lazard, D; Fernández-Tomás, C; Gariglio, P; Weinmann, R

    1989-01-01

    To further characterize the mechanism involved in poliovirus-induced inhibition of HeLa cells mRNA synthesis, in vitro formation of DNA-protein complexes between nuclear upstream stimulatory transcription factor (USF) and the adenovirus type 2 major late promoter upstream promoter element (UPE; located between -45 and -65 base pairs) was studied. Using the gel shift assay, we found differences between the UPE-protein complex formed with partially purified nuclear extracts from poliovirus-infected HeLa cells and that obtained in the presence of mock-infected extracts. Formation of the modified UPE-USF complex coincided with virus-induced inhibition of host cell RNA synthesis in vivo and with a less efficient in vitro transcriptional activity of the nuclear extracts from infected cells. Furthermore, using a cross-linking protocol, we found that the host 46-kilodalton UPE-binding USF factor was severely diminished and that a virus-induced or -modified 50-kilodalton polypeptide appeared to be specifically bound to the UPE template. Images PMID:2474675

  19. Seeing Beyond the Naked Eye in a Planetarium

    NASA Astrophysics Data System (ADS)

    Fairall, A.

    2005-12-01

    I have a philosophy that the traditional naked-eye sky, as usually shown in planetariums, should only be an introductory step in portraying the Universe. Consequently, over the years I have produced 'inter alia' various versions of an enhanced Milky Way (the latest based on Axel Mellenger's panorama), the extragalactic sky and the radio sky for projection on planetarium domes. I also put together a three-dimensional planetarium show-the audience being equipped with ChromDepth(tm) spectacles- which stepped from the Solar System to the cosmic microwave background. The advent of digital technology now makes all this much easier. Currently, Labyrinth, a visualization program developed in-house, serves much the same function as the Hayden Planetarium's Partiview, but also permits rendering and fl y-throughs of large-scale structures. It allows viewers to explore local cosmography. Labyrinth can produce images that operate with the 3-D spectacles; we have also produced a version of Partiview that does the same.

  20. The Role of Electron Microscopy in Studying the Continuum of Changes in Membranous Structures during Poliovirus Infection

    PubMed Central

    Rossignol, Evan D.; Yang, Jie E.; Bullitt, Esther

    2015-01-01

    Replication of the poliovirus genome is localized to cytoplasmic replication factories that are fashioned out of a mixture of viral proteins, scavenged cellular components, and new components that are synthesized within the cell due to viral manipulation/up-regulation of protein and phospholipid synthesis. These membranous replication factories are quite complex, and include markers from multiple cytoplasmic cellular organelles. This review focuses on the role of electron microscopy in advancing our understanding of poliovirus RNA replication factories. Structural data from the literature provide the basis for interpreting a wide range of biochemical studies that have been published on virus-induced lipid biosynthesis. In combination, structural and biochemical experiments elucidate the dramatic membrane remodeling that is a hallmark of poliovirus infection. Temporal and spatial membrane modifications throughout the infection cycle are discussed. Early electron microscopy studies of morphological changes following viral infection are re-considered in light of more recent data on viral manipulation of lipid and protein biosynthesis. These data suggest the existence of distinct subcellular vesicle populations, each of which serves specialized roles in poliovirus replication processes. PMID:26473912

  1. Inactivated poliovirus type 2 vaccine delivered to rat skin via high density microprojection array elicits potent neutralising antibody responses.

    PubMed

    Muller, David A; Pearson, Frances E; Fernando, Germain J P; Agyei-Yeboah, Christiana; Owens, Nick S; Corrie, Simon R; Crichton, Michael L; Wei, Jonathan C J; Weldon, William C; Oberste, M Steven; Young, Paul R; Kendall, Mark A F

    2016-01-01

    Polio eradication is progressing rapidly, and the live attenuated Sabin strains in the oral poliovirus vaccine (OPV) are being removed sequentially, starting with type 2 in April 2016. For risk mitigation, countries are introducing inactivated poliovirus vaccine (IPV) into routine vaccination programs. After April 2016, monovalent type 2 OPV will be available for type 2 outbreak control. Because the current IPV is not suitable for house-to-house vaccination campaigns (the intramuscular injections require health professionals), we developed a high-density microprojection array, the Nanopatch, delivered monovalent type 2 IPV (IPV2) vaccine to the skin. To assess the immunogenicity of the Nanopatch, we performed a dose-matched study in rats, comparing the immunogenicity of IPV2 delivered by intramuscular injection or Nanopatch immunisation. A single dose of 0.2 D-antigen units of IPV2 elicited protective levels of poliovirus antibodies in 100% of animals. However, animals receiving IPV2 by IM required at least 3 immunisations to reach the same neutralising antibody titres. This level of dose reduction (1/40th of a full dose) is unprecedented for poliovirus vaccine delivery. The ease of administration coupled with the dose reduction observed in this study points to the Nanopatch as a potential tool for facilitating inexpensive IPV for mass vaccination campaigns. PMID:26911254

  2. Isolation and Characterization of a Type 2 Vaccine-Derived Poliovirus from Environmental Surveillance in China, 2012

    PubMed Central

    Liu, Yao; Xu, Aiqiang; Lin, Xiaojuan; Yoshida, Hiromu; Xiong, Ping; Zhu, Shuangli; Wang, Suting; Yan, Dongmei; Song, Lizhi; Wang, Haiyan; Cui, Ning; Xu, Wenbo

    2013-01-01

    Environmental surveillance of poliovirus on sewage has been conducted in Shandong Province, China since 2008. A type 2 vaccine-derived poliovirus (VDPV) with 7 mutations in VP1 coding region was isolated from the sewage collected in the city of Jinan in December 2012. The complete genome sequencing analysis of this isolate revealed 25 nucleotide substitutions, 7 of which resulted in amino acid alteration. No evidence of recombination with other poliovirus serotypes was observed. The virus did not lose temperature sensitive phenotype at 40°C. An estimation based on the evolution rate of the P1 coding region suggested that evolution time of this strain might be 160–176 days. VP1 sequence analysis revealed that this VDPV strain is of no close relationship with other local type 2 polioviruses (n = 66) from sewage collected between May 2012 and June 2013, suggesting the lack of its circulation in the local population. The person who excreted the virus was not known and no closely related virus was isolated in local population via acute flaccid paralysis surveillance. By far this is the first report of VDPV isolated from sewage in China, and these results underscore the value of environmental surveillance in the polio surveillance system even in countries with high rates of OPV coverage. PMID:24386319

  3. Viral precursor protein P3 and its processed products perform discrete and essential functions in the poliovirus RNA replication complex

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The differential use of protein precursors and their products is a key strategy used during poliovirus replication. To characterize the role of protein precursors during replication, we examined the complementation profiles of mutants that inhibited 3D polymerase or 3C-RNA binding activity. We showe...

  4. Inactivated poliovirus type 2 vaccine delivered to rat skin via high density microprojection array elicits potent neutralising antibody responses

    PubMed Central

    Muller, David A.; Pearson, Frances E.; Fernando, Germain J.P.; Agyei-Yeboah, Christiana; Owens, Nick S.; Corrie, Simon R.; Crichton, Michael L.; Wei, Jonathan C.J.; Weldon, William C.; Oberste, M. Steven; Young, Paul R.; Kendall, Mark A. F.

    2016-01-01

    Polio eradication is progressing rapidly, and the live attenuated Sabin strains in the oral poliovirus vaccine (OPV) are being removed sequentially, starting with type 2 in April 2016. For risk mitigation, countries are introducing inactivated poliovirus vaccine (IPV) into routine vaccination programs. After April 2016, monovalent type 2 OPV will be available for type 2 outbreak control. Because the current IPV is not suitable for house-to-house vaccination campaigns (the intramuscular injections require health professionals), we developed a high-density microprojection array, the Nanopatch, delivered monovalent type 2 IPV (IPV2) vaccine to the skin. To assess the immunogenicity of the Nanopatch, we performed a dose-matched study in rats, comparing the immunogenicity of IPV2 delivered by intramuscular injection or Nanopatch immunisation. A single dose of 0.2 D-antigen units of IPV2 elicited protective levels of poliovirus antibodies in 100% of animals. However, animals receiving IPV2 by IM required at least 3 immunisations to reach the same neutralising antibody titres. This level of dose reduction (1/40th of a full dose) is unprecedented for poliovirus vaccine delivery. The ease of administration coupled with the dose reduction observed in this study points to the Nanopatch as a potential tool for facilitating inexpensive IPV for mass vaccination campaigns. PMID:26911254

  5. Improved thermal stability of oxide-supported naked gold nanoparticles by ligand-assisted pinning

    SciTech Connect

    Moreno, C; Divins, N. J.; Gazquez, Jaume; Varela, Maria; Angurell, I; Llorca, J

    2012-01-01

    We report a method to improve the thermal stability, up to 900 C, of bare-metal (naked) gold nanoparticles supported on top of SiO{sub 2} and SrTiO{sub 3} substrates via ligand-assisted pinning. This approach leads to monodisperse naked gold nanoparticles without significant sintering after thermal annealing in air at 900 C. The ligand-assisted pinning mechanism is described.

  6. Hypersensitivity to contact inhibition provides a clue to cancer resistance of naked mole-rat.

    PubMed

    Seluanov, Andrei; Hine, Christopher; Azpurua, Jorge; Feigenson, Marina; Bozzella, Michael; Mao, Zhiyong; Catania, Kenneth C; Gorbunova, Vera

    2009-11-17

    The naked mole-rat is the longest living rodent with a maximum lifespan exceeding 28 years. In addition to its longevity, naked mole-rats have an extraordinary resistance to cancer as tumors have never been observed in these rodents. Furthermore, we show that a combination of activated Ras and SV40 LT fails to induce robust anchorage-independent growth in naked mole-rat cells, while it readily transforms mouse fibroblasts. The mechanisms responsible for the cancer resistance of naked mole-rats were unknown. Here we show that naked mole-rat fibroblasts display hypersensitivity to contact inhibition, a phenomenon we termed "early contact inhibition." Contact inhibition is a key anticancer mechanism that arrests cell division when cells reach a high density. In cell culture, naked mole-rat fibroblasts arrest at a much lower density than those from a mouse. We demonstrate that early contact inhibition requires the activity of p53 and pRb tumor suppressor pathways. Inactivation of both p53 and pRb attenuates early contact inhibition. Contact inhibition in human and mouse is triggered by the induction of p27(Kip1). In contrast, early contact inhibition in naked mole-rat is associated with the induction of p16(Ink4a). Furthermore, we show that the roles of p16(Ink4a) and p27(Kip1) in the control of contact inhibition became temporally separated in this species: the early contact inhibition is controlled by p16(Ink4a), and regular contact inhibition is controlled by p27(Kip1). We propose that the additional layer of protection conferred by two-tiered contact inhibition contributes to the remarkable tumor resistance of the naked mole-rat. PMID:19858485

  7. Applications of the tunneling method to particle decay and radiation from naked singularities

    NASA Astrophysics Data System (ADS)

    di Criscienzo, Roberto; Vanzo, Luciano; Zerbini, Sergio

    2010-05-01

    Following recent literature on dS instability in presence of interactions, we study the decay of massive particles in general FRW models and the emission from naked singularities either associated with 4 D charged black holes or 2 D shock waves, by means of the Hamilton-Jacobi tunneling method. It is shown that the two-dimensional semiclassical tunneling amplitude from a naked singularity computed in that way is the same as the one-loop result of quantum field theory.

  8. H1PVAT is a novel and potent early-stage inhibitor of poliovirus replication that targets VP1.

    PubMed

    Tijsma, Aloys; Thibaut, Hendrik Jan; Spieser, Stéphane A H; De Palma, Armando; Koukni, Mohamed; Rhoden, Eric; Oberste, Steve; Pürstinger, Gerhard; Volny-Luraghi, Antonia; Martin, Javier; Marchand, Arnaud; Chaltin, Patrick; Neyts, Johan; Leyssen, Pieter

    2014-10-01

    A novel small molecule, H1PVAT, was identified as a potent and selective inhibitor of the in vitro replication of all three poliovirus serotypes, whereas no activity was observed against other enteroviruses. Time-of-drug-addition studies revealed that the compound interfered with an early stage of virus replication. Four independently-selected H1PVAT-resistant virus variants uniformly carried the single amino acid substitution I194F in the VP1 capsid protein. Poliovirus type 1 strain Sabin, reverse-engineered to contain this substitution, proved to be completely insensitive to the antiviral effect of H1PVAT and was cross-resistant to the capsid-binding inhibitors V-073 and pirodavir. The VP1 I194F mutant had a smaller plaque phenotype than wild-type virus, and the amino acid substitution rendered the virus more susceptible to heat inactivation. Both for the wild-type and VP1 I194F mutant virus, the presence of H1PVAT increased the temperature at which the virus was inactivated, providing evidence that the compound interacts with the viral capsid, and that capsid stabilization and antiviral activity are not necessarily correlated. Molecular modeling suggested that H1PVAT binds with high affinity in the pocket underneath the floor of the canyon that is involved in receptor binding. Introduction of the I194F substitution in the model of VP1 induced a slight concerted rearrangement of the core β-barrel in this pocket, which disfavors binding of the compound. Taken together, the compound scaffold, to which H1PVAT belongs, may represent another promising class of poliovirus capsid-binding inhibitors next to V-073 and pirodavir. Potent antivirals against poliovirus will be essential in the poliovirus eradication end-game. PMID:25043639

  9. The compatibility of inactivated-Enterovirus 71 vaccination with Coxsackievirus A16 and Poliovirus immunizations in humans and animals

    PubMed Central

    Mao, Qunying; Wang, Yiping; Shao, Jie; Ying, Zhifang; Gao, Fan; Yao, Xin; Li, Changgui; Ye, Qiang; Xu, Miao; Li, Rongcheng; Zhu, Fengcai; Liang, Zhenglun

    2015-01-01

    Enterovirus 71 (EV71) is the key pathogen for Hand, Foot, and Mouth Disease (HFMD) and can result in severe neurological complications and death among young children. Three inactivated-EV71 vaccines have gone through phase III clinical trials and have demonstrated good safety and efficacy. These vaccines will benefit young children under the threat of severe HFMD. However, the potential immunization-related compatibility for different enterovirus vaccines remains unclear, making it hard to include the EV71 vaccine in Expanded Program on Immunization (EPI). Here, we measured the neutralizing antibodies (NTAbs) against EV71, Coxsackievirus A16 (CA16) and Poliovirus from infants enrolled in those EV71 vaccine clinical trials. The results indicated that the levels of NTAb GMTs for EV71 increased significantly in all 3 vaccine groups (high, middle and low dosages, respectively) post-vaccination. Seroconversion ratios and Geometric mean fold increase were significantly higher in the vaccine groups (≥7/9 and 8.9~228.1) than in the placebo group (≤1/10 and 0.8~1.7, P < 0.05). But no similar NTAb response trends were found in CA16 and 3 types of Poliovirus. The decrease of 3 types of Poliovirus NTAb GMTs and an increase of CA16 GMTs post-EV71-vaccination were found in vaccine and placebo groups. Further animal study on CA16 and poliovirus vaccine co-immunization or pre-immunization with EV71 vaccine in mice indicated that there was no NTAb cross-activity between EV71 and CA16/Poliovirus. Our research showed that inactivated-EV71 vaccine has good specific-neutralizing capacity and can be included in EPI. PMID:25715318

  10. Environmental Surveillance of Polioviruses in Rio de Janeiro, Brazil, in Support to the Activities of Global Polio Eradication Initiative.

    PubMed

    de Oliveira Pereira, Joseane Simone; da Silva, Lidiane Rodrigues; de Meireles Nunes, Amanda; de Souza Oliveira, Silas; da Costa, Eliane Veiga; da Silva, Edson Elias

    2016-03-01

    Wild polioviruses still remain endemic in three countries (Afghanistan, Pakistan, and Nigeria) and re-emergency of wild polio has been reported in previously polio-free countries. Environmental surveillance has been used as a supplementary tool in monitoring the circulation of wild poliovirus (PVs) and/or vaccine-derived PVs even in the absence of acute flaccid paralysis cases. This study aimed to monitor the presence of polioviruses in wastewater samples collected at one wastewater treatment plant located in the municipality of Rio de Janeiro, Brazil. From December 2011 to June 2012 and from September to December 2012, 31 samples were collected and processed. RD and L20B cell cultures were able to isolate PVs and non-polio enteroviruses in 27/31 samples. Polioviruses were isolated in eight samples (type 1 Sabin = 1, type 2 Sabin = 5, and type 3 Sabin = 2). Vaccine-derived polioviruses were not detected nor evidence of recombination with other PVs or non-polio enterovirus serotypes were observed among the isolates. The Sabin-related serotypes 2 and 3 presented nucleotide substitutions in positions associated with the neurovirulent phenotype at the 5'-UTR. Changes in important Amino acid residues at VP1 were also observed in the serotypes 2 and 3. Environmental surveillance has been used successfully in monitoring the circulation of PVs and non-polio enteroviruses and it is of crucial importance in the final stages of the WHO global polio eradication initiative. Our results show the continuous circulation of Sabin-like PVs and non-polio enteroviruses in the analyzed area during the study period. PMID:26538420

  11. Seeing protein monolayers with naked eye through plasmonic Fano resonances.

    PubMed

    Yanik, Ahmet A; Cetin, Arif E; Huang, Min; Artar, Alp; Mousavi, S Hossein; Khanikaev, Alexander; Connor, John H; Shvets, Gennady; Altug, Hatice

    2011-07-19

    We introduce an ultrasensitive label-free detection technique based on asymmetric Fano resonances in plasmonic nanoholes with far reaching implications for point-of-care diagnostics. By exploiting extraordinary light transmission phenomena through high-quality factor (Q(solution) ∼ 200) subradiant dark modes, we experimentally demonstrate record high figures of merits (FOMs as high as 162) for intrinsic detection limits surpassing that of the gold standard prism coupled surface-plasmon sensors (Kretschmann configuration). Our experimental record high sensitivities are attributed to the nearly complete suppression of the radiative losses that are made possible by the high structural quality of the fabricated devices as well as the subradiant nature of the resonances. Steep dispersion of the plasmonic Fano resonance profiles in high-quality plasmonic sensors exhibit dramatic light intensity changes to the slightest perturbations within their local environment. As a spectacular demonstration of the extraordinary sensitivity and the quality of the fabricated biosensors, we show direct detection of a single monolayer of biomolecules with naked eye using these Fano resonances and the associated Wood's anomalies. To fabricate high optical-quality sensors, we introduce a high-throughput lift-off free evaporation fabrication technique with extremely uniform and precisely controlled nanofeatures over large areas, leading to resonance line-widths comparable to that of the ideally uniform structures as confirmed by our time-domain simulations. The demonstrated label-free sensing platform offers unique opportunities for point-of-care diagnostics in resource poor settings by eliminating the need for fluorescent labeling and optical detection instrumentation (camera, spectrometer, etc.) as well as mechanical and light isolation. PMID:21715661

  12. Poliovirus Polyuridylic Acid Polymerase and RNA Replicase Have the Same Viral Polypeptide

    PubMed Central

    Flanegan, James B.; Baltimore, David

    1979-01-01

    A poliovirus-specific polyuridylic acid [poly(U)] polymerase that copies a polyadenylic acid template complexed to an oligouridylic acid primer was isolated from the membrane fraction of infected HeLa cells and was found to sediment at 4 to 5S on a linear 5 to 20% glycerol gradient. When the poly(U) polymerase was isolated from cells labeled with [35S]methionine and was analyzed by glycerol gradient centrifugation and polyacrylamide gel electrophoresis, the position of only one viral protein was found to correlate with the location of enzyme activity. This protein had an apparent molecular weight of 62,500 based on its electrophoretic mobility relative to that of several molecular weight standards and was designated p63. When the poly(U) polymerase was isolated from the soluble fraction of a cytoplasmic extract, the activity was found to sediment at about 7S. In this case, however, both p63 and NCVP2 (77,000-dalton precursor of p63) cosedimented with the 7S activity peak. When the 7S polymerase activity was purified by phosphocellulose chromatography, both p63 and NCVP2 were found to co-chromatograph with poly(U) polymerase activity. The poliovirus replicase complexed with its endogenous RNA template was isolated from infected cells labeled with [35S]methionine and was centrifuged through a linear 15 to 30% glycerol gradient. The major viral polypeptide component in a 26S peak of replicase activity was p63, but small amounts of other poliovirus proteins were also present. When the replicase-template complex was treated with RNase T1 before centrifugation, a single peak of activity was found that sedimented at 20S and contained only labeled p63. Thus, p63 was found to be the only viral polypeptide in the replicase bound to its endogenous RNA template, and appears to be active as a poly(U) polymerase either as a monomer protein or as a 7S complex. Images PMID:219230

  13. Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

    PubMed Central

    Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

    2015-01-01

    ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

  14. [Circulating vaccine-derived poliovirus type 2 outbreak in Democratic Republic of Congo 2011-2012].

    PubMed

    Bazira, L; Coulibaly, T; Mayenga, M; Ncharre, C; Yogolelo, R; Mbule, A; Moudzeo, H; Lwamba, P; Mulumba, A W; Cabore, J

    2015-10-01

    According to the WHO records of 2013, the incidence of poliomyelitis was reduced by more than 99%, the number of endemic countries decreased from 125 in 1988 to 3 in 2013 and over 10 million cases were prevented from poliomyelitis thanks to the intensive use of Oral polio vaccine (OPV). However, the emergence of circulating vaccine-derived poliovirus strains (cVDPV), causing serious epidemics like the wild poliovirus, is a major challenge on the final straight towards the goal of eradication and OPV cessation. This paper describes the cVDPVoutbreak that occurred in the Democratic Republic of Congo (DRC) from November 2011 to April 2012. All children under 15 years of age with acute flaccid paralysis (AFP) and confirmed presence of cVDPV in the stool samples were included. Thirty (30) children, all from the administrative territories of Bukama and Malemba Nkulu in the Katanga Province (south-east DRC), were reported. The virus responsible was the cVDPV type 2 (0.7% -3.5% divergent from the reference Sabin 2 strain) in 29 children (97%) and the ambiguous vaccine-derived poliovirus strain (0.7% divergent) was confirmed in one case (3%), a boy seventeen months old and already vaccinated four times with OPV. Twentyfive children (83%) were protected by any of the routine EPI vaccines and 3 children (10%) had never received any dose of OPV. In reaction, DRC has conducted five local campaigns over a period of 10 months (from January to October 2012) and the epidemic was stopped after the second round performed in March 2012. As elsewhere in similar conditions, low immunization coverage, poor sanitation conditions and the stop of the use of OPV2 have favoured the emergence of the third cVDPV epidemic in DRC. The implementation of the Strategic Plan for Polio eradication and endgame strategic plan 2013-2018 will prevent the emergence of cVDPV and set up the conditions for a coordinated OPV phase out. PMID:26288132

  15. Host range of poliovirus is restricted to simians because of a rapid sequence change of the poliovirus receptor gene during evolution.

    PubMed

    Ida-Hosonuma, M; Sasaki, Y; Toyoda, H; Nomoto, A; Gotoh, O; Yonekawa, H; Koike, S

    2003-01-01

    The host range of most poliovirus (PV) strains is restricted to simians. This host range specificity is believed to be determined by the interaction between PV and its receptor molecule. To elucidate the molecular basis of this species-specific infection of PV, we cloned orthologs of the PV receptor (PVR) gene ( pvr) as well as those of PV receptor-related genes 1 and 2 ( prr1 and prr2) from various mammalian species. These three genes are widely present in mammalian genomes including those of non-susceptible species. Comparison of the deduced amino acid sequences of PVR orthologs revealed that the NH(2)-terminal immunoglobulin-like domain (domain 1), which is the virus binding site in the human PVR, is highly variable among species, whereas that of PRR1 is highly conserved. Domain 1 of the PVR orthologs for the ring-tailed lemur and rabbit, which are not susceptible to PV, show only 51 and 61% amino acid sequence identity to that of human PVR, respectively. Chimeric PVR proteins that have the domain 1 of the ring-tailed lemur and rabbit PVRs failed to serve as receptors for PV. These results suggest that rapid changes in the domain 1 sequence during mammalian evolution determined the host range restriction of PV. PMID:12536294

  16. Seroprevalence of poliovirus antibodies among 7-month-old infants after 4 doses of oral polio vaccine in Sistan-va-Baluchestan, Islamic Republic of Iran.

    PubMed

    Izadi, S; Shahmahmoodi, S; Zahraei, S M; Dorostkar, F; Majdzadeh, S-R

    2015-02-01

    Despite high coverage rates of polio vaccine in the Islamic Republic of Iran, the seroconversion rates of infants may be inadequate. This study measured seroprevalence of antibodies against poliovirus serotypes 1 to 3 (PV1, PV2 and PV3) in 7-month-old infants who had received at least 4 doses of trivalent oral polio vaccine. A serosurvey was conducted in 2010 in rural areas of Chabahar, Sistan-va-Baluchestan province. Using cluster sampling, 72 eligible infants were tested for antibody against the 3 poliovirus serotypes according to WHO guidelines. Antibody titres ≥ 1:10 were considered positive. The seropositive rates for antibody against PV1, PV2 and PV3 were 84.7%, 95.8% and 70.8% respectively. Only 63.9% of participants were seropositive for antibodies against all 3 poliovirus serotypes. Except for PV2, the seroprevalence of antibody against the other 2 poliovirus serotypes, especially PV3, was unsatisfactory. PMID:25876819

  17. Comparison of ozone inactivation, in flowing water, of hepatitis A virus, poliovirus 1, and indicator organisms

    SciTech Connect

    Herbold, K.; Flehmig, B.; Botzenhart, K. )

    1989-11-01

    In steadily flowing water at 20 degrees C and pH 7, five organisms had the following order of resistance to ozone (at constant levels of ozone): poliovirus 1 (PV1) less than Escherichia coli less than hepatitis A virus (HAV) less than Legionella pneumophila serogroup 6 less than Bacillus subtilis spores. The tests were repeated at 10 degrees C with HAV, PV1, and E. coli. Ozone inactivation of HAV and E. coli was faster at 10 degrees C than at 20 degrees C. At 20 degrees C, 0.25 to 0.38 mg of O3 per liter was required for complete inactivation of HAV but only 0.13 mg of O3 per liter was required for complete inactivation of PV1.

  18. Effects of actinomycin D on the cytopathology induced by poliovirus in HEp-2 cells.

    PubMed

    Guskey, L E; Wolff, D A

    1974-11-01

    One possible mechanism of virus-induced cell damage is that the redistributed (released) lysosomal enzymes produce the cytopathic effect during cytolytic types of infections such as poliovirus in HEp-2 cells. To determine if the lysosomal enzyme redistribution and cell damage are host-cell directed, we studied sensitivity of these events to the action of actinomycin D. By the use of actinomycin D at concentrations producing the least toxicity but maximal effectiveness in shuting down cell RNA synthesis, it was shown that the cytopathic effect and enzyme redistribution were not inhibited and, therefore, not directly controlled and induced by the cell genome in response to the virus infection. Evaluation of cytopathic effect by a phase contrast microscopy method detected changes earlier than the erythrocin B uptake method. PMID:4372396

  19. Effects of Actinomycin D on the Cytopathology Induced by Poliovirus in HEp-2 Cells

    PubMed Central

    Guskey, Louis E.; Wolff, David A.

    1974-01-01

    One possible mechanism of virus-induced cell damage is that the redistributed (released) lysosomal enzymes produce the cytopathic effect during cytolytic types of infections such as poliovirus in HEp-2 cells. To determine if the lysosomal enzyme redistribution and cell damage are host-cell directed, we studied sensitivity of these events to the action of actinomycin D. By the use of actinomycin D at concentrations producing the least toxicity but maximal effectiveness in shuting down cell RNA synthesis, it was shown that the cytopathic effect and enzyme redistribution were not inhibited and, therefore, not directly controlled and induced by the cell genome in response to the virus infection. Evaluation of cytopathic effect by a phase contrast microscopy method detected changes earlier than the erythrocin B uptake method. PMID:4372396

  20. Interim CDC guidance for polio vaccination for travel to and from countries affected by wild poliovirus.

    PubMed

    Wallace, Gregory S; Seward, Jane F; Pallansch, Mark A

    2014-07-11

    In the prevaccine era, infection with wild poliovirus (WPV) was common worldwide, with seasonal peaks and epidemics in the summer and fall in temperate areas. The incidence of poliomyelitis in the United States declined rapidly after the licensure of inactivated polio vaccine (IPV) in 1955 and live oral polio vaccine (OPV) in the 1960s. The last cases of indigenously acquired WPV in the United States occurred in 1979, the last WPV case in a U.S. resident traveling abroad occurred in 1986, and the last WPV imported case was in 1993. Since 2000, the United States has exclusively used IPV, resulting in prevention of 8-10 vaccine-associated paralytic poliomyelitis cases annually. In 2005, an unvaccinated U.S. adult traveling abroad acquired vaccine-associated paralytic poliomyelitis after contact with an infant recently vaccinated with OPV. PMID:25006826

  1. Human-Mouse Hybrid Cell Lines and Susceptibility to Poliovirus 1

    PubMed Central

    Wang, Richard; Pollack, Robert; Kusano, Toshihisa; Green, Howard

    1970-01-01

    A number of human-mouse somatic hybrid cell lines have been prepared, containing from 3 to 12 human biarmed chromosomes. These lines were susceptible to poliovirus type 1, producing viral yields comparable to those of the human parental cells. A small proportion of the cells of these lines survived the polio infection, and their progeny were solidly resistant to reinfection with the virus. Both sensitive and resistant hybrids produced virus following infection with viral ribonucleic acid, indicating that the cytoplasm of the resistant hybrids was able to support viral multiplication. Viral adsorption studies carried out at 4 C showed that the resistant sublines had negligible ability to adsorb the virus. It was concluded that the hybrid cells became resistant to polio through loss of the human chromosome bearing the gene for the receptor substance. PMID:4317112

  2. Immunogenicity of poliovirus vaccines in chronically malnourished infants: A randomized controlled trial in Pakistan

    PubMed Central

    Saleem, Ali Faisal; Mach, Ondrej; Quadri, Farheen; Khan, Asia; Bhatti, Zaid; Rehman, Najeeb ur; Zaidi, Sohail; Weldon, William C.; Oberste, Steven M.; Salama, Maha; Sutter, Roland W.; Zaidi, Anita K.M.

    2015-01-01

    Reaching high population immunity against polioviruses (PV) is essential to achieving global polio eradication. Efficacy of oral poliovirus vaccine (OPV) varies and is lower among children living in tropical areas with impoverished environments. Malnutrition found as a risk factor for lower serological protection against PV. We compared whether inactivated polio vaccine (IPV) can be used to rapidly close the immunity gap among chronically malnourished (stunted) infants in Pakistan who will not be eligible for the 14 week IPV dose in routine EPI schedule. A phase 3, multicenter 4-arm randomized controlled trial conducted at five Primary Health Care (PHC) centers in Karachi, Pakistan. Infants, 9–12 months were stratified by length for age Z score into chronically malnourished and normally nourished. Infants were randomized to receive one dose of either bivalent OPV (bOPV) alone or bOPV + IPV. Baseline seroprevalence of PV antibodies and serum immune response to study vaccine dose were assessed by neutralization assay. Vaccine PV shedding in stool was evaluated 7 days after a bOPV challenge dose. Sera and stool were analyzed from 852/928 (92%) enrolled children. At baseline, the seroprevalence was 85.6% (n = 386), 73.6% (n = 332), and 70.7% (n = 319) in malnourished children against PV types 1, 2 and 3 respectively; and 94.1% (n = 448), 87.0% (n = 441) and 83.6% (n = 397) in the normally nourished group (p < 0.05). Children had previously received 9–10 doses of bOPV (80%) or tOPV (20%). One dose of IPV + bOPV given to malnourished children increased their serological protection (PV1, n = 201, 97.6%; PV2, n = 198, 96.1% and PV3, n = 189, 91.7%) to parity with normally nourished children who had not received IPV (p = <0.001). Seroconversion and boosting for all three serotypes was significantly more frequent in children who received IPV + bOPV than in those with bOPV only (p < 0.001) in both strata. Shedding of polioviruses in stool did

  3. Molecular Characterization and Phylogenetic Relationship of Wild Type 1 Poliovirus Strains Circulating across Pakistan and Afghanistan Bordering Areas during 2010–2012

    PubMed Central

    Shaukat, Shahzad; Angez, Mehar; Alam, Muhammad Masroor; Sharif, Salmaan; Khurshid, Adnan; Malik, Farzana; Rehman, Lubna; Zaidi, Syed Sohail Zahoor

    2014-01-01

    Pakistan and Afghanistan share a long uncontrolled border with extensive population movement on both sides. Wild poliovirus transmission has never been interrupted in this block due to war against terrorism, poor public health infrastructure, misconceptions about polio vaccines and inadequate immunization activities. All these issues complicate the eradication operations and reinforce the complexity of wiping out poliomyelitis from this region. This study illustrates the origins and routes of cross-border wild poliovirus type 1 (WPV1) transmission during 2010–2012 between Pakistan and Afghanistan. Sequence analyses were conducted based on complete VP1 capsid protein sequences for WPV1 study strains to determine the origin of poliovirus genetic lineages and their evolutionary relationships. Phylogenetic tree was constructed from VP1 gene sequences applying Maximum Likelihood method using Kimura 2- parameter model in MEGA program v 5.0. A total of 72 (14.3%) out of 502 wild-type 1 polioviruses were found circulating in border areas of both countries during 2010–2012. Molecular phylogenetic analysis classified these strains in to two sub-genotypes with four clusters and 18 lineages. Genetic data confirmed that the most of WPV1 lineages (12; 66.6%) were transmitted from Pakistan to Afghanistan. However, the genetic diversity was significantly reduced during 2012 as most of the lineages were completely eliminated. In conclusion, Pakistan-Afghanistan block has emerged as a single poliovirus reservoir sharing the multiple poliovirus lineages due to uncontrolled movement of people across the borders between two countries. If it is neglected, it can jeopardize the extensive global efforts done so-far to eradicate the poliovirus infection. Our data will be helpful to devise the preventive strategies for effective control of wild poliovirus transmission in this region. PMID:25229826

  4. Novel diversity-oriented synthesis-derived respiratory syncytial virus inhibitors identified via a high throughput replicon-based screen.

    PubMed

    Duvall, Jeremy R; VerPlank, Lynn; Ludeke, Barbara; McLeod, Sarah M; Lee, Maurice D; Vishwanathan, Karthick; Mulrooney, Carol A; Le Quement, Sebastian; Yu, Qin; Palmer, Michelle A; Fleming, Paul; Fearns, Rachel; Foley, Michael A; Scherer, Christina A

    2016-07-01

    Respiratory syncytial virus (RSV) infections affect millions of children and adults every year. Despite the significant disease burden, there are currently no safe and effective vaccines or therapeutics. We employed a replicon-based high throughput screen combined with live-virus triaging assays to identify three novel diversity-oriented synthesis-derived scaffolds with activity against RSV. One of these small molecules is shown to target the RSV polymerase (L protein) to inhibit viral replication and transcription; the mechanisms of action of the other small molecules are currently unknown. The compounds described herein may provide attractive inhibitors for lead optimization campaigns. PMID:27059228

  5. Transformation of microorganisms with the plasmid vector with the replicon from pAC1 from Acetobacter pasteurianus.

    PubMed

    Grones, J; Turna, J

    1995-01-26

    A number of gram-negative and gram-positive bacteria species was screened for the expression of the gram-negative plasmid pACK5 and pACT72 with replicon of pAC1 plasmid from Acetobacter pasteurianus. As was described previously, both plasmids were expressed in Escherichia coli, Acetobacter pasteurianus, Acetobacter aceti, Shigella spp. and Citrobacter spp. Expressions of plasmids were successful in twelve species tested, Comamonas terrigena, Salmonella typhimurium, Serratia marcescens, Bacillus cereus, Bacillus megatericum, Bacillus subtilis, Lactobacillus helveticus, Micrococcus luteus, Sarcina lutea, Staphylococcus aureus, Staphylococcus epidermidis, Streptoccocus feacalis, and the stability of plasmid DNA was tested after cultivation in non-selective conditions. PMID:7832808

  6. Detection and quantification of poliovirus infection using FTIR spectroscopy and cell culture

    PubMed Central

    2011-01-01

    Background In a globalized word, prevention of infectious diseases is a major challenge. Rapid detection of viable virus particles in water and other environmental samples is essential to public health risk assessment, homeland security and environmental protection. Current virus detection methods, especially assessing viral infectivity, are complex and time-consuming, making point-of-care detection a challenge. Faster, more sensitive, highly specific methods are needed to quantify potentially hazardous viral pathogens and to determine if suspected materials contain viable viral particles. Fourier transform infrared (FTIR) spectroscopy combined with cellular-based sensing, may offer a precise way to detect specific viruses. This approach utilizes infrared light to monitor changes in molecular components of cells by tracking changes in absorbance patterns produced following virus infection. In this work poliovirus (PV1) was used to evaluate the utility of FTIR spectroscopy with cell culture for rapid detection of infective virus particles. Results Buffalo green monkey kidney (BGMK) cells infected with different virus titers were studied at 1 - 12 hours post-infection (h.p.i.). A partial least squares (PLS) regression method was used to analyze and model cellular responses to different infection titers and times post-infection. The model performs best at 8 h.p.i., resulting in an estimated root mean square error of cross validation (RMSECV) of 17 plaque forming units (PFU)/ml when using low titers of infection of 10 and 100 PFU/ml. Higher titers, from 103 to 106 PFU/ml, could also be reliably detected. Conclusions This approach to poliovirus detection and quantification using FTIR spectroscopy and cell culture could potentially be extended to compare biochemical cell responses to infection with different viruses. This virus detection method could feasibly be adapted to an automated scheme for use in areas such as water safety monitoring and medical diagnostics. PMID

  7. Characterization of Poliovirus Neutralization Escape Mutants of Single-Domain Antibody Fragments (VHHs)

    PubMed Central

    Schotte, Lise; Thys, Bert; Strauss, Mike; Filman, David J.; Rombaut, Bart

    2015-01-01

    To complete the eradication of poliovirus and to protect unvaccinated people subsequently, the development of one or more antiviral drugs will be necessary. A set of five single-domain antibody fragments (variable parts of the heavy chain of a heavy-chain antibody [VHHs]) with an in vitro neutralizing activity against poliovirus type 1 was developed previously (B. Thys, L. Schotte, S. Muyldermans, U. Wernery, G. Hassanzadeh-Ghassabeh, and B. Rombaut, Antiviral Res 87:257–264, 2010, http://dx.doi.org/10.1016/j.antiviral.2010.05.012), and their mechanisms of action have been studied (L. Schotte, M. Strauss, B. Thys, H. Halewyck, D. J. Filman, M. Bostina, J. M. Hogle, and B. Rombaut, J Virol 88:4403–4413, 2014, http://dx.doi.org/10.1128/JVI.03402-13). In this study, neutralization escape mutants were selected for each VHH. Sequencing of the P1 region of the genome showed that amino acid substitutions are found in the four viral proteins of the capsid and that they are located both in proximity to the binding sites of the VHHs and in regions further away from the canyon and hidden beneath the surface. Characterization of the mutants demonstrated that they have single-cycle replication kinetics that are similar to those of their parental strain and that they are all drug (VHH) independent. Their resistant phenotypes are stable, as they do not regain full susceptibility to the VHH after passage over HeLa cells in the absence of VHH. They are all at least as stable as the parental strain against heat inactivation at 44°C, and three of them are even significantly (P < 0.05) more resistant to heat inactivation. The resistant variants all still can be neutralized by at least two other VHHs and retain full susceptibility to pirodavir and 35-1F4. PMID:26014941

  8. Antiviral Activity of Trichilia catigua Bark Extracts for Herpesvirus and Poliovirus.

    PubMed

    Espada, Samantha F; Faccin-Galhardi, Ligia C; Rincao, Vinicius P; Bernardi, Ana L S; Lopes, Nayara; Longhini, Renata; de Mello, Joao C P; Linhares, Rosa E C; Nozawa, Carlos

    2015-01-01

    Herpesvirus and poliovirus are responsible for important diseases in human and animal. Trichilia catigua a Brazilian native plant known as catiguá has several medicinal properties among them antimicrobial for bacteria and protozoa, however, no antiviral activity has been reported yet. This study evaluated the antiviral activity of the crude extract (CE) and aqueous and ethyl acetate fractions (AF, EAF) obtained from T. catigua in the replication of the Herpes simplex virus (HSV-1), bovine herpesvirus (BoHV-1) and poliovirus (PV-1). The cytotoxicity was analyzed by MTT assay and the antiviral effect was determined by the addition of extracts (0.25 to 100.0 μg/ml), before (-2h and -1h), during (Oh) and after (1h and 2h) the viral infection, by plaque reduction assay, in HEp-2 cell culture. The virucidal activity and inhibition of viral adsorption were also evaluated. In addition, the combination index (CI) with Acyclovir (ACV - reference drug) was determined for HSV-1. CE, AF and EAF showed a low toxicity (CC(50) >400 µg/ml) and low inhibitory concentration (IC50), ranging from 2.44-34.25 µg/ml for herpesvirus and 0.67 to 1.8 µg/ml for PV-1, associated with high selectivity index. The tested compounds showed high virucidal effect and high ability to inhibit viral adsorption, for all virus. The CI demonstrated a synergic effect (CI<1) for AF and EAF comparatively to acyclovir (ACV). Our study demonstrated that the extract and fractions of T. catigua is promising for future antiviral drug design with economically feasible production. PMID:25941883

  9. Limited and localized outbreak of newly emergent type 2 vaccine-derived poliovirus in Sichuan, China.

    PubMed

    Yan, Dongmei; Zhang, Yong; Zhu, Shuangli; Chen, Na; Li, Xiaolei; Wang, Dongyan; Ma, Xiaozhen; Zhu, Hui; Tong, Wenbin; Xu, Wenbo

    2014-07-01

    From August 2011 to February 2012, an outbreak caused by type 2 circulating vaccine-derived poliovirus (cVDPV) occurred in Aba County, Sichuan, China. During the outbreak, four type 2 VDPVs (≥0.6% nucleotide divergence in the VP1 region relative to the Sabin 2 strain) were isolated from 3 patients with acute flaccid paralysis (AFP) and one close contact. In addition, a type 2 pre-VDPV (0.3% to 0.5% divergence from Sabin 2) that was genetically related to these type 2 VDPVs was isolated from another AFP patient. These 4 patients were all unimmunized children 0.7 to 1.1 years old. Nucleotide sequencing revealed that the 4 VDPV isolates differed from Sabin 2 by 0.7% to 1.2% in nucleotides in the VP1 region and shared 5 nucleotide substitutions with the pre-VDPV. All 5 isolates were closely related, and all were S2/S3/S2/S3 recombinants sharing common recombination crossover sites. Although the two major determinants of attenuation and temperature sensitivity phenotype of Sabin 2 (A481 in the 5' untranslated region and Ile143 in the VP1 protein) had reverted in all 5 isolates, one VDPV (strain CHN16017) still retained the temperature sensitivity phenotype. Phylogenetic analysis of the third coding position of the complete P1 coding region suggested that the cVDPVs circulated locally for about 7 months following the initiating oral poliovirus vaccine (OPV) dose. Our findings reinforce the point that cVDPVs can emerge and spread in isolated communities with immunity gaps and highlight the emergence risks of type 2 cVDPVs accompanying the trivalent OPV used. To solve this issue, it is recommended that type 2 OPV be removed from the trivalent OPV or that inactivated polio vaccine (IPV) be used instead. PMID:24850620

  10. Co-Circulation and Evolution of Polioviruses and Species C Enteroviruses in a District of Madagascar

    PubMed Central

    Rakoto-Andrianarivelo, Mala; Guillot, Sophie; Iber, Jane; Balanant, Jean; Blondel, Bruno; Riquet, Franck; Martin, Javier; Kew, Olen; Randriamanalina, Bakolalao; Razafinimpiasa, Lalatiana; Rousset, Dominique; Delpeyroux, Francis

    2007-01-01

    Between October 2001 and April 2002, five cases of acute flaccid paralysis (AFP) associated with type 2 vaccine-derived polioviruses (VDPVs) were reported in the southern province of the Republic of Madagascar. To determine viral factors that favor the emergence of these pathogenic VDPVs, we analyzed in detail their genomic and phenotypic characteristics and compared them with co-circulating enteroviruses. These VDPVs appeared to belong to two independent recombinant lineages with sequences from the type 2 strain of the oral poliovaccine (OPV) in the 5′-half of the genome and sequences derived from unidentified species C enteroviruses (HEV-C) in the 3′-half. VDPV strains showed characteristics similar to those of wild neurovirulent viruses including neurovirulence in poliovirus-receptor transgenic mice. We looked for other VDPVs and for circulating enteroviruses in 316 stools collected from healthy children living in the small area where most of the AFP cases occurred. We found vaccine PVs, two VDPVs similar to those found in AFP cases, some echoviruses, and above all, many serotypes of coxsackie A viruses belonging to HEV-C, with substantial genetic diversity. Several coxsackie viruses A17 and A13 carried nucleotide sequences closely related to the 2C and the 3Dpol coding regions of the VDPVs, respectively. There was also evidence of multiple genetic recombination events among the HEV-C resulting in numerous recombinant genotypes. This indicates that co-circulation of HEV-C and OPV strains is associated with evolution by recombination, resulting in unexpectedly extensive viral diversity in small human populations in some tropical regions. This probably contributed to the emergence of recombinant VDPVs. These findings give further insight into viral ecosystems and the evolutionary processes that shape viral biodiversity. PMID:18085822

  11. pUCL287 plasmid from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315 represents a new theta-type replicon family of lactic acid bacteria.

    PubMed

    Benachour, A; Frère, J; Novel, G

    1995-05-01

    A cryptic plasmid, pUCL287, was isolated from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315. It had a theta-type mechanism of replication in its natural host. Its minimal replicon, Rep287, was isolated on a 1.6-kb EcoRI fragment. The Rep287 host range included the genera Pediococcus, Enterococcus, Lactobacillus and Leuconostoc but not genus Lactococcus. Plasmids hybridizing to pUCL287 are rare among lactic acid bacteria. As assessed by hybridization, Rep287 is dissimilar to pAM beta 1, pIP501 and pUCL22, representatives of the most common theta-type replicon groups in Gram-positive bacteria. Therefore, pUCL287 appears to represent a new theta-type replicon family from lactic acid bacteria. PMID:7750734

  12. Alphavirus replicon-based adjuvants enhance the immunogenicity and effectiveness of Fluzone ® in rhesus macaques.

    PubMed

    Carroll, Timothy D; Matzinger, Shannon R; Barro, Mario; Fritts, Linda; McChesney, Michael B; Miller, Christopher J; Johnston, Robert E

    2011-01-29

    Venezuelan equine encephalitis virus replicon particles (VRP) without a transgene (null VRP) have been used to adjuvant effective humoral [1], cellular [2], and mucosal [3] immune responses in mice. To assess the adjuvant activity of null VRP in the context of a licensed inactivated influenza virus vaccine, rhesus monkeys were immunized with Fluzone(®) alone or Fluzone(®) mixed with null VRP and then challenged with a human seasonal influenza isolate, A/Memphis/7/2001 (H1N1). Compared to Fluzone(®) alone, Fluzone(®)+null VRP immunized animals had stronger influenza-specific CD4(+) T cell responses (4.4 fold) with significantly higher levels of virus-specific IFN-γ (7.6 fold) and IL-2 (5.3 fold) producing CD4+ T cells. Fluzone(®)+null VRP immunized animals also had significantly higher plasma anti-influenza IgG (p<0.0001, 1.3 log) and IgA (p<0.05, 1.2 log) levels. In fact, the mean plasma anti-influenza IgG titers after one Fluzone(®)+null VRP immunization was 1.2 log greater (p<0.04) than after two immunizations with Fluzone(®) alone. After virus challenge, only Fluzone(®)+null VRP immunized monkeys had a significantly lower level of viral replication (p<0.001) relative to the unimmunized control animals. Although little anti-influenza antibody was detected in the respiratory secretions after immunization, strong anamnestic anti-influenza IgG and IgA responses were present in secretions of the Fluzone(®)+null VRP immunized monkeys immediately after challenge. There were significant inverse correlations between influenza RNA levels in tracheal lavages and plasma anti-influenza HI and IgG anti-influenza antibody titers prior to challenge. These results demonstrate that null VRP dramatically improve both the immunogenicity and protection elicited by a licensed inactivated influenza vaccine. PMID:21111777

  13. A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

    PubMed

    Sánchez-Puig, Juana M; Lorenzo, María M; Blasco, Rafael

    2013-01-01

    Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses. PMID:24130722

  14. Naked mole rats exhibit metabolic but not ventilatory plasticity following chronic sustained hypoxia.

    PubMed

    Chung, Danielle; Dzal, Yvonne A; Seow, Allison; Milsom, William K; Pamenter, Matthew E

    2016-03-30

    Naked mole rats are among the most hypoxia-tolerant mammals identified and live in chronic hypoxia throughout their lives. The physiological mechanisms underlying this tolerance, however, are poorly understood. Most vertebrates hyperventilate in acute hypoxia and exhibit an enhanced hyperventilation following acclimatization to chronic sustained hypoxia (CSH). Conversely, naked mole rats do not hyperventilate in acute hypoxia and their response to CSH has not been examined. In this study, we explored mechanisms of plasticity in the control of the hypoxic ventilatory response (HVR) and hypoxic metabolic response (HMR) of freely behaving naked mole rats following 8-10 days of chronic sustained normoxia (CSN) or CSH. Specifically, we investigated the role of the major inhibitory neurotransmitter γ-amino butyric acid (GABA) in mediating these responses. Our study yielded three important findings. First, naked mole rats did not exhibit ventilatory plasticity following CSH, which is unique among adult animals studied to date. Second, GABA receptor (GABAR) antagonism altered breathing patterns in CSN and CSH animals and modulated the acute HVR in CSN animals. Third, naked mole rats exhibited GABAR-dependent metabolic plasticity following long-term hypoxia, such that the basal metabolic rate was approximately 25% higher in normoxic CSH animals than CSN animals, and GABAR antagonists modulated this increase. PMID:27009224

  15. Venezuelan Equine Encephalitis Virus Replicon Particle Vaccine Protects Nonhuman Primates from Intramuscular and Aerosol Challenge with Ebolavirus

    PubMed Central

    Herbert, Andrew S.; Kuehne, Ana I.; Barth, James F.; Ortiz, Ramon A.; Nichols, Donald K.; Zak, Samantha E.; Stonier, Spencer W.; Muhammad, Majidat A.; Bakken, Russell R.; Prugar, Laura I.; Olinger, Gene G.; Groebner, Jennifer L.; Lee, John S.; Pratt, William D.; Custer, Max; Kamrud, Kurt I.; Smith, Jonathan F.; Hart, Mary Kate

    2013-01-01

    There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine. PMID:23408633

  16. Replicon typing of plasmids carrying blaCTX-M-1 in Enterobacteriaceae of animal, environmental and human origin

    PubMed Central

    Zurfluh, Katrin; Jakobi, Gianna; Stephan, Roger; Hächler, Herbert; Nüesch-Inderbinen, Magdalena

    2014-01-01

    Objectives: The aim of this work was to determine the plasmid replicon profiles of a collection of blaCTX-M-1-positive enterobacterial strains. The isolates originated from chicken in the production pyramid, healthy food-producing animals at slaughter (chicken, calves, and pigs), chicken retail meat, environmental isolates originating from water bodies, and isolates from humans. A selection of IncI and IncN plasmids were characterized by multilocus sequence typing in order to determine their epidemiological relatedness. Methods: Transconjugants of 74 blaCTX-M-1-positive isolates were analyzed by PCR-based replicon typing and by PCR-based plasmid multilocus sequence typing. Results: The incompatibility groups detected among the blaCTX-M-1-harboring plasmids included IncI1, IncN, IncHI1B, IncF, IncFIIS, IncFIB, and IncB/O, with plasmid lineage IncI1/ST3 predominating in isolates from chicken and from humans. Lineage IncN/ST1 was detected mainly in isolates from pigs. For the first time, blaCTX-M-1 genes encoded on IncHI1 plasmids were detected in isolates from cattle and from water bodies. Conclusions: This study identifies plasmid lineages that are contributing to the dissemination of blaCTX-M-1 genes in the food chain, the environment, and humans. PMID:25400623

  17. Virus Replication, Cytopathology, and Lysosomal Enzyme Response of Mitotic and Interphase Hep-2 Cells Infected with Poliovirus

    PubMed Central

    Bienz, Kurt; Egger, Denise; Wolff, David A.

    1973-01-01

    Mitotic Hep-2 cells, selected by the PEL (colloidal silica) density gradient method and held in mitosis with Colcemid, are readily infected by poliovirus type I (Mahoney). They produce and release the same amount of virus as interphase, random-growing cells. In contrast to interphase cells, mitotic cells show no detectable virus-induced cytopathic effect at the light microscopy level and only slight alterations, consisting of small clusters of vacuoles, at the electron microscopy level. Mitotic cells contain the same total amount of lysosomal enzymes per cell as interphase cells, but they display no redistribution of lysosomal enzymes during the virus infection as interphase cells do. This supports the view that lysosomal enzyme redistribution is associated with the cytopathic effect in poliovirus infection but shows that virus synthesis and release is not dependent on either the cytopathic effect or lysosomal enzyme release. The possible reasons for the lack of cytopathic effect in mitotic cells are discussed. Images PMID:4121707

  18. Development and consideration of global policies for managing the future risks of poliovirus outbreaks: insights and lessons learned through modeling.

    PubMed

    Thompson, Kimberly M; Duintjer Tebbens, Radboud J; Pallansch, Mark A; Kew, Olen M; Sutter, Roland W; Aylward, R Bruce; Watkins, Margaret; Gary, Howard; Alexander, James P; Venczel, Linda; Johnson, Denise; Cáceres, Victor M; Sangrujee, Nalinee; Jafari, Hamid; Cochi, Stephen L

    2006-12-01

    The success of the Global Polio Eradication Initiative promises to bring large benefits, including sustained improvements in quality of life (i.e., cases of paralytic disease and deaths avoided) and costs saved from cessation of vaccination. Obtaining and maintaining these benefits requires that policymakers manage the transition from the current massive use of oral poliovirus vaccine (OPV) to a world without OPV and free of the risks of potential future reintroductions of live polioviruses. This article describes the analytical journey that began in 2001 with a retrospective case study on polio risk management and led to development of dynamic integrated risk, economic, and decision analysis tools to inform global policies for managing the risks of polio. This analytical journey has provided several key insights and lessons learned that will be useful to future analysts involved in similar complex decision-making processes. PMID:17184398

  19. Combined use of inactivated and oral poliovirus vaccines in refugee camps and surrounding communities - Kenya, December 2013.

    PubMed

    Sheikh, Mohamed A; Makokha, Frederick; Hussein, Abdullahi M; Mohamed, Gedi; Mach, Ondrej; Humayun, Kabir; Okiror, Samuel; Abrar, Leila; Nasibov, Orkhan; Burton, John; Unshur, Ahmed; Wannemuehler, Kathleen; Estivariz, Concepcion F

    2014-03-21

    Since the launch of the Global Polio Eradication Initiative (GPEI) in 1988, circulation of indigenous wild poliovirus (WPV) has continued without interruption in only three countries: Afghanistan, Nigeria, and Pakistan. During April-December 2013, a polio outbreak caused by WPV type 1 (WPV1) of Nigerian origin resulted in 217 cases in or near the Horn of Africa, including 194 cases in Somalia, 14 cases in Kenya, and nine cases in Ethiopia (all cases were reported as of March 10, 2014). During December 14-18, 2013, Kenya conducted the first-ever campaign providing inactivated poliovirus vaccine (IPV) together with oral poliovirus vaccine (OPV) as part of its outbreak response. The campaign targeted 126,000 children aged ≤59 months who resided in Somali refugee camps and surrounding communities near the Kenya-Somalia border, where most WPV1 cases had been reported, with the aim of increasing population immunity levels to ensure interruption of any residual WPV transmission and prevent spread from potential new importations. A campaign evaluation and vaccination coverage survey demonstrated that combined administration of IPV and OPV in a mass campaign is feasible and can achieve coverage >90%, although combined IPV and OPV campaigns come at a higher cost than OPV-only campaigns and require particular attention to vaccinator training and supervision. Future operational studies could assess the impact on population immunity and the cost-effectiveness of combined IPV and OPV campaigns to accelerate interruption of poliovirus transmission during polio outbreaks and in certain areas in which WPV circulation is endemic. PMID:24647400

  20. The MTSU Uranidrome: A Naked-Eye Observatory for Teaching Astronomy and Geometry

    NASA Astrophysics Data System (ADS)

    Klumpe, Eric W.

    2006-12-01

    Prior to the invention of the telescope astronomy was done with the naked eye. The astronomical models that ancient astronomers were able to construct based upon their naked-eye observations are impressive. Following those examples, Middle Tennessee State University has built a 30-meter diameter, naked-eye, observatory to serve as a hands-on teaching tool for astronomy and geometry. The primary goal is for astronomy students to use their own measurements to develop accurate models of how our solar system works and to better appreciate the creativity of ancient astronomers. Typical Uranidrome activities include measuring the axial tilt of the Earth, the latitude and longitude of MTSU, the spin rate and orbital rate of the Earth, the circumference of the Earth, true-north relative to magnetic-north, short-term predictions of eclipses, and using the Sun to determine the date and time. The Uranidrome was funded by grants from NASA and Middle Tennessee State University.

  1. The naked T Tauri stars - The low-mass pre-main sequence unveiled

    NASA Technical Reports Server (NTRS)

    Walter, Frederick M.

    1987-01-01

    The search for low-mass premain-sequence (PMS) stars associated with X-ray sources in regions of star formation is discussed. The survey to date has revealed at least 30 low-mass PMS stars in the Tau-Aur region, and a comparable number in Oph. These stars are the naked T Tau stars, unveiled versions of the well-known classical T Tau stars. The properties of these newly discovered PMS stars and their relation to the classical T Tau stars are discussed, and it is concluded that the naked T Tau stars are the true low-mass PMS stars, and that the observable characteristics defining the classical T Tau stars are due to the interaction of an underlying, fairly normal star with a dominant circumstellar environment. The impact the naked T Tau stars are likely to have on models of the PMS evolution of low-mass stars is considered.

  2. Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1

    PubMed Central

    Belov, George A.; Kovtunovych, Gennadiy; Jackson, Catherine L.; Ehrenfeld, Ellie

    2010-01-01

    Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the Arf GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to BFA, an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism. PMID:20497182

  3. [Poliovirus uptake into and excretion from oysters: a model experiment for elimination of Norwalk-like viruses from oysters].

    PubMed

    Fukuta, Miwa; Kawada, Kazunobu; Yano, Takuya; Sugiyama, Akira; Nakayama, Osamu; Nishio, Osamu; Sekine, Hiromasa; Sakurai, Nakao

    2003-02-01

    Outbreaks of gastroenteritis caused by Norwalk-like viruses are often induced by the consumption of raw shellfish such as oysters. Incidences reach a peak during the cold season in Japan, when seawater temperatures fall below 10 degrees C. We investigated oysters' uptake and excretion of viruses, over varying lengths of exposure, monitoring the effects of changes in temperature and flow rate of seawater, and the presence of plankton. The study was performed using a poliovirus and an experimental circulatory system, which was framed on the same principle as a model practically used for the depuration of oysters. Polioviruses present in the seawater were taken rapidly into the midgut gland of oysters. However, virus levels detected in oysters at both 10 degrees C and 20 degrees C were decreased to approximately 1/1,000 to 1/10,000 within 6 hrs after the circulatory seawater was replaced by UV irradiated seawater. These results demonstrate the effectiveness of the circulatory depuration system for the elimination of poliovirus from oysters, and indicate that controlling the temperature and flow rate of the circulatory system could decrease the risk of NLV infection. PMID:12661085

  4. Expression and subcellular localization of poliovirus VPg-precursor protein 3AB in eukaryotic cells: evidence for glycosylation in vitro.

    PubMed Central

    Datta, U; Dasgupta, A

    1994-01-01

    The poliovirus-encoded, membrane-associated VPg-precursor polypeptide 3AB has been implicated in the initiation of viral RNA synthesis. We have expressed 3AB and 3A polypeptides in eukaryotic cells and examined their localization using indirect immunofluorescence and a direct in vitro membrane-binding assay. Results presented here demonstrate that both 3AB and 3A are capable of localizing in the endoplasmic reticulum and the Golgi apparatus in transfected HeLa cells in the absence of any other poliovirus protein. We have also shown that the carboxy-terminal 18 amino acids of 3A that constitute an amphipathic domain are important in membrane binding of 3A and 3AB. Additionally, we demonstrate that a significant fraction of both 3A and 3AB can be glycosylated in a membrane-dependent fashion during in vitro translation in reticulocyte lysate. We demonstrate that 6-diazo-5-oxo-L-norleucine, an inhibitor of glycoprotein synthesis, significantly inhibits poliovirus RNA synthesis in vivo. The implications of glycosylation of 3AB (and 3A) in viral replication are discussed. Images PMID:8207820

  5. Observational distinction between black holes and naked singularities: the role of the redshift function

    NASA Astrophysics Data System (ADS)

    Ortiz, Néstor; Sarbach, Olivier; Zannias, Thomas

    2015-12-01

    We suggest that the redshift of photons traveling from past to future null infinity through a collapsing object could provide an observational signature capable of differentiating between the formation of a globally naked singularity and the formation of an event horizon. Supporting evidence for this idea is drawn from the analysis of photons with zero angular momentum through the center of a collapsing spherical dust cloud. We show that the frequency shift as a function of proper time with respect to stationary observers has distinct features depending on whether the object collapses to a black hole or a naked singularity.

  6. Solution-based nanosensors for in-field detection with the naked eye.

    PubMed

    Paterson, S; de la Rica, R

    2015-05-21

    Nanomaterials are revolutionising analytical applications with low-cost tests that enable detecting a target molecule in a few steps and with the naked eye. With this approach, non-experts can perform analyses on-site and without utilising electronic readers. This is advantageous in point-of-care diagnostics, in-field measurements and analyses performed in resource-constrained settings. Here we review the main strategies adopted for detecting analytes with the naked eye and at the point of need using plasmonic nanosensors, catalytic nanoparticles and fluorescent nanomaterials. Examples of the detection of ions, glucose, small molecules, peptides and proteins with the nanosensors are explained in detail. PMID:25802896

  7. Test bodies and naked singularities: is the self-force the cosmic censor?

    PubMed

    Barausse, Enrico; Cardoso, Vitor; Khanna, Gaurav

    2010-12-31

    Jacobson and Sotiriou showed that rotating black holes could be spun up past the extremal limit by the capture of nonspinning test bodies, if one neglects radiative and self-force effects. This would represent a violation of the cosmic censorship conjecture in four-dimensional, asymptotically flat spacetimes. We show that for some of the trajectories giving rise to naked singularities, radiative effects can be neglected. However, for these orbits the conservative self-force is important, and seems to have the right sign to prevent the formation of naked singularities. PMID:21231640

  8. The RepA_N replicons of Gram-positive bacteria: a family of broadly distributed but narrow host range plasmids

    PubMed Central

    Weaver, Keith E.; Kwong, Stephen M.; Firth, Neville; Francia, Maria Victoria

    2009-01-01

    The pheromone-responsive conjugative plasmids of Enterococcus faecalis and the multi-resistance plasmids pSK1 and pSK41 of Staphylococcus aureus are among the best studied plasmids native to Gram-positive bacteria. Although these plasmids seem largely restricted to their native hosts, protein sequence comparison of their replication initiator proteins indicates that they are clearly related. Homology searches indicate that these replicons are representatives of a large family of plasmids and a few phage that are widespread among the low G+C Gram-positive bacteria. We propose to name this family the RepA_N family of replicons after the annotated conserved domain that the initiator protein contains. Detailed sequence comparisons indicate that the initiator protein phylogeny is largely congruent with that of the host, suggesting that the replicons have evolved along with their current hosts and that intergeneric transfer has been rare. However, related proteins were identified on chromosomal regions bearing characteristics indicative of ICE elements, and the phylogeny of these proteins displayed evidence of more frequent intergeneric transfer. Comparison of stability determinants associated with the RepA_N replicons suggests that they have a modular evolution as has been observed in other plasmid families. PMID:19100285

  9. Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells.

    PubMed

    Wehbe, Michel; Huguenin, Antoine; Leveque, Nicolas; Semler, Bert L; Hamze, Monzer; Andreoletti, Laurent; Bouin, Alexis

    2016-04-01

    Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10-20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral replication activities. Our objectives were to generate a reporter replicon from a cardiotropic prototype CV-B3/28 strain and to characterize its replication properties into human cardiac primary cells. To obtain this replicon, a cDNA plasmid containing the full CV-B3/28 genome flanked by a hammerhead ribozyme sequence and an MluI restriction site was generated and used as a platform for the insertion of sequences encoding emerald green fluorescent protein (EmGFP) in place of those encoding VP3. In vitro transcribed RNA from this plasmid was transfected into HeLa cells and human primary cardiac cells and was able to produce EmGFP and VP1-containing polypeptides. Moreover, non-structural protein biological activity was assessed by the specific cleavage of eIF4G1 by viral 2A(pro). Viral RNA replication was indirectly demonstrated by inhibition assays, fluoxetine was added to cell culture and prevented the EmGFP synthesis. Our results indicated that the EmGFP CV-B3 replicon was able to replicate and translate as well as the CV-B3/28 prototype strain. Our EmGFP CV-B3 replicon will be a valuable tool to readily investigate CV-B3 replication activities in human target cell models. PMID:26800776

  10. Construction of an infectious molecular clone of Japanese encephalitis virus genotype V and its derivative subgenomic replicon capable of expressing a foreign gene.

    PubMed

    Ishikawa, Tomohiro; Abe, Makoto; Masuda, Michiaki

    2015-01-01

    Japanese encephalitis virus (JEV) genotype V was originally isolated in Malaysia in 1952 and has long been restricted to the area. In 2009, sudden emergence of the genotype V in China and Korea was reported, suggesting expansion of its geographical distribution. Although studies on the genotype V are becoming more important, they have been limited partly due to lack of its infectious molecular clone. In this study, a plasmid carrying cDNA corresponding to the entire genome of JEV Muar strain, which belongs to genotype V, in the downstream of T7 promoter was constructed. Electroporation of viral RNA transcribed by T7 RNA polymerase (T7RNAP) in vitro from the plasmid led to production of progeny viruses both in mammalian and mosquito cells. Also, transfection of the infectious clone plasmid into mammalian cells expressing T7RNAP transiently or stably was demonstrated to generate infectious progenies. When the viral structural protein genes were partially deleted from the full-length cDNA, the subgenomic RNA transcribed in vitro from the modified plasmid was shown to replicate itself in mammalian cells as a replicon. The replicon carrying the firefly luciferase gene in place of the deleted structural protein genes was also shown to efficiently replicate itself and express luciferase in mammalian cells. Compared with the replicon derived from JEV genotype III (Nakayama strain), the genotype V-derived replicon appeared to be more tolerant to introduction of a foreign gene. The infectious clone and the replicons constructed in this study may serve as useful tools for characterizing JEV genotype V. PMID:25451067

  11. Development of "Naked-Tufted" Seed Coat Mutants for Potential Use in Cotton Production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Use of chemical mutagenesis has been highly successful in most major crops but has only recently been used in improving cotton. The objective of this research was to develop ‘naked-tufted’ seed mutants and to incorporate this genetic trait into cotton to enhance crop quality and reduce processing co...

  12. Registration of tufted-naked seed in upland cotton germplasm 9023n4t

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A naked-tufted mutant called 9023n4t (PI 667553) was developed from the cultivar SC 9023 (Gossypium hirsutum L.) through chemical mutagenesis. This germplasm was developed by the Department of Plant and Soil Science, Texas Tech University and released in April, 2013. This mutant is quite unique sinc...

  13. Newtonian analogue of static general relativistic spacetimes: An extension to naked singularities

    NASA Astrophysics Data System (ADS)

    Ghosh, Shubhrangshu; Sarkar, Tamal; Bhadra, Arunava

    2015-10-01

    We formulate a generic Newtonian-like analogous potential for static spherically symmetric general relativistic (GR) spacetime and subsequently derived proper Newtonian-like analogous potential corresponding to Janis-Newman-Winicour (JNW) and Reissner-Nordström (RN) spacetimes, both exhibiting naked singularities. The derived potentials were found to reproduce the entire GR features including the orbital dynamics of the test particle motion and the orbital trajectories, with precise accuracy. The nature of the particle orbital dynamics including their trajectory profiles in JNW and RN geometries show altogether different behaviors with distinctive traits as compared to the nature of particle dynamics in Schwarzschild geometry. Exploiting the Newtonian-like analogous potentials, we found that the radiative efficiency of a geometrically thin and optically thick Keplerian accretion disk around naked singularities corresponding to both JNW and RN geometries, in general, is always higher than that for Schwarzschild geometry. The derived potentials would thus be useful to study astrophysical processes, especially to investigate more complex accretion phenomena in active galactic nuclei (AGNs) or in x-ray binaries (XRBs) in the presence of naked singularities and thereby to explore any noticeable differences in their observational features from those in the presence of black holes (BHs) to ascertain outstanding debatable issues relating to gravity—whether the end state of gravitational collapse in our physical Universe renders BH or naked singularity.

  14. Virtually naked: virtual environment reveals sex-dependent nature of skin disclosure.

    PubMed

    Lomanowska, Anna M; Guitton, Matthieu J

    2012-01-01

    The human tendency to reveal or cover naked skin reflects a competition between the individual propensity for social interactions related to sexual appeal and interpersonal touch versus climatic, environmental, physical, and cultural constraints. However, due to the ubiquitous nature of these constraints, isolating on a large scale the spontaneous human tendency to reveal naked skin has remained impossible. Using the online 3-dimensional virtual world of Second Life, we examined spontaneous human skin-covering behavior unhindered by real-world climatic, environmental, and physical variables. Analysis of hundreds of avatars revealed that virtual females disclose substantially more naked skin than virtual males. This phenomenon was not related to avatar hypersexualization as evaluated by measurement of sexually dimorphic body proportions. Furthermore, analysis of skin-covering behavior of a population of culturally homogeneous avatars indicated that the propensity of female avatars to reveal naked skin persisted despite explicit cultural norms promoting less revealing attire. These findings have implications for further understanding how sex-specific aspects of skin disclosure influence human social interactions in both virtual and real settings. PMID:23300580

  15. Notoedres muris infestation in a naked-tailed rat Melomys cervinipes.

    PubMed

    McKenzie, R A; Green, P E; Knox, E

    1976-10-01

    Large numbers of Notoedres muris were present in thick crusty lesions on both ears, the nose, one forelimb and tail-base of a free-living naked-tailed rat, Melomys cervinipes, on North Stradbroke Island, Queensland, Australia. The source of infestation was obscure. PMID:16502682

  16. Blunted Behavioral and C Fos Responses to Acidic Fumes in the African Naked Mole-Rat

    PubMed Central

    LaVinka, Pamela Colleen; Park, Thomas J.

    2012-01-01

    Acidosis in the skin triggers activation of pain pathways and behaviors indicative of pain in vertebrates. The exception is the naked mole-rat, the only known vertebrate to show physiological and behavioral insensitivity to acid pain in the skin. The goal of the present study was to determine behavioral and physiological responses of this species to airborne acidic fumes, which would be expected to affect the trigeminal pain pathway in other species. Behaviorally, naked mole-rats did not avoid fumes from moderately high concentrations of acetic acid (10 and 20%), and c Fos labeling showed no increase in activity in the trigeminal nuclei and nucleus tractus solitarius. In contrast, these concentrations triggered behavioral aversion and increased Fos activity in other laboratory rodents. For a very high concentration of acetic acid (50%), naked mole-rats showed significant avoidance behavior and increased Fos labeling in the nucleus tractus solitarius caudal region, which receives vagal chemosensory information. However, there was no increase in trigeminal labeling, and in fact, activity significantly decreased. This pattern is opposite of that associated with another irritant, ammonia fumes, which elicited an increase in trigeminal but not nucleus tractus solitarius Fos labeling, and no behavioral avoidance. Behavioral avoidance of acidic fumes, but no increased labeling in the trigeminal pain nucleus is consistent with the notion of adaptations to blunt acid pain, which would be advantageous for naked mole-rats as they normally live under chronically high levels of acidosis-inducing CO2. PMID:23028761

  17. Virtually Naked: Virtual Environment Reveals Sex-Dependent Nature of Skin Disclosure

    PubMed Central

    Lomanowska, Anna M.; Guitton, Matthieu J.

    2012-01-01

    The human tendency to reveal or cover naked skin reflects a competition between the individual propensity for social interactions related to sexual appeal and interpersonal touch versus climatic, environmental, physical, and cultural constraints. However, due to the ubiquitous nature of these constraints, isolating on a large scale the spontaneous human tendency to reveal naked skin has remained impossible. Using the online 3-dimensional virtual world of Second Life, we examined spontaneous human skin-covering behavior unhindered by real-world climatic, environmental, and physical variables. Analysis of hundreds of avatars revealed that virtual females disclose substantially more naked skin than virtual males. This phenomenon was not related to avatar hypersexualization as evaluated by measurement of sexually dimorphic body proportions. Furthermore, analysis of skin-covering behavior of a population of culturally homogeneous avatars indicated that the propensity of female avatars to reveal naked skin persisted despite explicit cultural norms promoting less revealing attire. These findings have implications for further understanding how sex-specific aspects of skin disclosure influence human social interactions in both virtual and real settings. PMID:23300580

  18. Status of the GENIUS-Test-Facility with Naked Germanium Detectors in Liquid Nitrogen

    NASA Astrophysics Data System (ADS)

    Klapdor-Kleingrothaus, H. V.; Krivosheina, I. V.

    2007-08-01

    GENIUS-TF-II is a setup of six naked high purity Ge detectors (15 kg) in liquid nitrogen in Gran Sasso. It has been installed in October, 2004 - after the first four naked Ge detectors had been installed on May 5, 2003 (GENIUS-TF-I). The GENIUS-Test-Facility (GENIUS-TF) is the first and up to now only setup ever testing the novel technique aiming at extreme background reduction in search for rare decays in particular underground. The goal of GENIUS-TF was to test some key operational parameters of the full GENIUS project proposal in 1997.1,2,3,4,5,6 Simultaneous physical goal is to search for the annual modulation of the Dark Matter signal.10,23 After operation of GENIUS-TF over three years with finally six naked Ge detectors (15 kg) in liquid nitrogen in Gran Sasso we realize the following problems: 1. Background from 222Rn diffusing into the setup, on a level far beyond the expectation. 2. Limited long-term stability of naked detectors in liquid nitrogen as result of increasing leakage current. None of the six detectors is running after three years with the nominal leakage current. Three of the six detectors do not work any more at all. These findings require further investigation on the way to a full-size GENIUS-like project.

  19. A Kunjin Replicon Virus-like Particle Vaccine Provides Protection Against Ebola Virus Infection in Nonhuman Primates.

    PubMed

    Pyankov, Oleg V; Bodnev, Sergey A; Pyankova, Olga G; Solodkyi, Vladislav V; Pyankov, Stepan A; Setoh, Yin Xiang; Volchkova, Valentina A; Suhrbier, Andreas; Volchkov, Viktor V; Agafonov, Alexander A; Khromykh, Alexander A

    2015-10-01

    The current unprecedented outbreak of Ebola virus (EBOV) disease in West Africa has demonstrated the urgent need for a vaccine. Here, we describe the evaluation of an EBOV vaccine candidate based on Kunjin replicon virus-like particles (KUN VLPs) encoding EBOV glycoprotein with a D637L mutation (GP/D637L) in nonhuman primates. Four African green monkeys (Cercopithecus aethiops) were injected subcutaneously with a dose of 10(9) KUN VLPs per animal twice with an interval of 4 weeks, and animals were challenged 3 weeks later intramuscularly with 600 plaque-forming units of Zaire EBOV. Three animals were completely protected against EBOV challenge, while one vaccinated animal and the control animal died from infection. We suggest that KUN VLPs encoding GP/D637L represent a viable EBOV vaccine candidate. PMID:25732811

  20. Crystal structure of CD155 and electron microscopic studies of its complexes with polioviruses

    SciTech Connect

    Zhang, Ping; Mueller, Steffen; Morais, Marc C.; Bator, Carol M.; Bowman, Valorie D.; Hafenstein, Susan; Wimmer, Eckard; Rossmann, Michael G.

    2010-11-02

    When poliovirus (PV) recognizes its receptor, CD155, the virus changes from a 160S to a 135S particle before releasing its genome into the cytoplasm. CD155 is a transmembrane protein with 3 Ig-like extracellular domains, D1-D3, where D1 is recognized by the virus. The crystal structure of D1D2 has been determined to 3.5-{angstrom} resolution and fitted into {approx}8.5-{angstrom} resolution cryoelectron microscopy reconstructions of the virus-receptor complexes for the 3 PV serotypes. These structures show that, compared with human rhinoviruses, the virus-receptor interactions for PVs have a greater dependence on hydrophobic interactions, as might be required for a virus that can inhabit environments of different pH. The pocket factor was shown to remain in the virus during the first recognition stage. The present structures, when combined with earlier mutational investigations, show that in the subsequent entry stage the receptor moves further into the canyon when at a physiological temperature, thereby expelling the pocket factor and separating the viral subunits to form 135S particles. These results provide a detailed analysis of how a nonenveloped virus can enter its host cell.

  1. Molecular tectonic model of virus structural transitions: the putative cell entry states of poliovirus.

    PubMed

    Belnap, D M; Filman, D J; Trus, B L; Cheng, N; Booy, F P; Conway, J F; Curry, S; Hiremath, C N; Tsang, S K; Steven, A C; Hogle, J M

    2000-02-01

    Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at approximately 22-A resolution by cryo-electron microscopy. The 135S and 80S particles are both approximately 4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 A were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell. PMID:10627545

  2. Poliovirus receptor (CD155) regulates a step in transendothelial migration between PECAM and CD99.

    PubMed

    Sullivan, David P; Seidman, Michael A; Muller, William A

    2013-03-01

    The movement of leukocytes across endothelium [referred to as diapedesis or transendothelial migration (TEM)] is a critical step in the inflammatory process. Recently, it was demonstrated that treatment of endothelial cells and monocytes with antibodies against poliovirus receptor (PVR; CD155) and DNAX-associated molecule-1 (DNAM-1; CD226) arrested monocytes over endothelial junctions and prevented TEM, suggesting that these molecules are involved in diapedesis. However, nothing was known about the mechanism by which PVR and DNAM-1 work in TEM. Herein, we show that, similar to endothelial PECAM interacting with leukocyte PECAM, activation of endothelial PVR with anti-PVR antibodies or interaction with its ligand, DNAM-1, results in recruitment of the tyrosine phosphatase Shp-2, and this process is dependent on Src kinases. Furthermore, differential and sequential treatment with blocking antibodies directed against PVR, DNAM-1, PECAM, and CD99 showed that endothelial PVR and monocyte DNAM-1 interact at and regulate a step between those regulated by PECAM and CD99. Further studies demonstrate that PVR resides in the recently identified lateral border recycling compartment, similar to PECAM and CD99. These findings suggest that the localization of adhesion/signaling molecules to the lateral border recycling compartment and the recruitment of Shp-2 may be common mechanisms for the regulation of TEM by endothelial cells. PMID:23333754

  3. Membrane Requirements for Uridylylation of the Poliovirus VPg Protein and Viral RNA Synthesis In Vitro

    PubMed Central

    Fogg, Mark H.; Teterina, Natalya L.; Ehrenfeld, Ellie

    2003-01-01

    Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation. PMID:14557626

  4. Analysis of mutations in oral poliovirus vaccine by hybridization with generic oligonucleotide microchips.

    SciTech Connect

    Proudnikov, D.; Kirillov, E.; Chumakov, K.; Donion, J.; Rezapkin, G.; Mirzabekov, A.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology; Center for Biologics Evaluation and Research

    2000-01-01

    This paper describes use of a new technology of hybridization with a micro-array of immobilized oligonucleotides for detection and quantification of neurovirulent mutants in Oral Poliovirus Vaccine (OPV). We used a micro-array consisting of three-dimensional gel-elements containing all possible hexamers (total of 4096 probes). Hybridization of fluorescently labelled viral cDNA samples with such microchips resulted in a pattern of spots that was registered and quantified by a computer-linked CCD camera, so that the sequence of the original cDNA could be deduced. The method could reliably identify single point mutations, since each of them affected fluorescence intensity of 12 micro-array elements. Micro-array hybridization of DNA mixtures with varying contents of point mutants demonstrated that the method can detect as little as 10% of revertants in a population of vaccine virus. This new technology should be useful for quality control of live viral vaccines, as well as for other applications requiring identification and quantification of point mutations.

  5. Cellular COPII Proteins Are Involved in Production of the Vesicles That Form the Poliovirus Replication Complex

    PubMed Central

    Rust, René C.; Landmann, Lukas; Gosert, Rainer; Tang, Bor Luen; Hong, Wanjin; Hauri, Hans-Peter; Egger, Denise; Bienz, Kurt

    2001-01-01

    Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway. PMID:11559814

  6. Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation

    PubMed Central

    Andreev, Dmitri E.; Hirnet, Juliane; Terenin, Ilya M.; Dmitriev, Sergey E.; Niepmann, Michael; Shatsky, Ivan N.

    2012-01-01

    Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5′ end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNAGly anticodon stem–loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus–host cell interaction are discussed. PMID:22373920

  7. Inhibition of host cell protein synthesis by UV-inactivated poliovirus.

    PubMed Central

    Helentjaris, T; Ehrenfeld, E

    1977-01-01

    The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell. Images PMID:189067

  8. WHO collaborative studies on poliovirus type 3 strains isolated during the 1968 poliomyelitis epidemic in Poland.

    PubMed

    Melnick, J L; Berencsi, G; Biberi-Moroeanu, S; Combiescu, A A; Furesz, J; Kantoch, M; Kostrzewski, J; Magrath, D I; Perkins, F T; Vonka, V; Cockburn, W C; Dömök, I; Assaad, F A

    1972-01-01

    In 1968 in Poland an extensive outbreak of poliomyelitis, caused by type 3 poliovirus, began about four months after small vaccine trials with the Leon 12a(1)b (Sabin) and USOL-D bac vaccine strains had been carried out. Because of the temporal association, and because the first cases appeared in the province in which the USOL-D vaccine trial was carried out, a detailed investigation of the strains isolated from cases in the epidemic was made in four laboratories in an attempt to determine whether they were related to the two vaccine strains or to a "wild" strain. All the studies were made under code. The rct marker was of no help in determining the relationship of the epidemic strains to the vaccine strains. The McBride test and the elution marker test clearly separated the Leon 12a(1)b strains from those from the cases, but were incapable of detecting whether the epidemic strains were related to the USOL-D bac strain or to wild type 3 strains. Thus the studies did not provide valid information on the origin of the epidemic. PMID:4346582

  9. An Introduction to Poliovirus: Pathogenesis, Vaccination, and the Endgame for Global Eradication.

    PubMed

    Minor, Philip D

    2016-01-01

    Poliomyelitis is caused by poliovirus, which is a positive strand non-enveloped virus that occurs in three distinct serotypes (1, 2, and 3). Infection is mainly by the fecal-oral route and can be confined to the gut by antibodies induced either by vaccine, previous infection or maternally acquired. Vaccines include the live attenuated strains developed by Sabin and the inactivated vaccines developed by Salk; the live attenuated vaccine (Oral Polio Vaccine or OPV) has been the main tool in the Global Program of Polio eradication of the World Health Organisation. Wild type 2 virus has not caused a case since 1999 and type 3 since 2012 and eradication seems near. However most infections are entirely silent so that sophisticated environmental surveillance may be needed to ensure that the virus has been eradicated, and the live vaccine can sometimes revert to virulent circulating forms under conditions that are not wholly understood. Cessation of vaccination is therefore an increasingly important issue and inactivated polio vaccine (IPV) is playing a larger part in the end game. PMID:26983727

  10. Molecular Tectonic Model of Virus Structural Transitions: the Putative Cell Entry States of Poliovirus

    PubMed Central

    Belnap, David M.; Filman, David J.; Trus, Benes L.; Cheng, Naiqian; Booy, Frank P.; Conway, James F.; Curry, Stephen; Hiremath, Chaitanya N.; Tsang, Simon K.; Steven, Alasdair C.; Hogle, James M.

    2000-01-01

    Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at ∼22-Å resolution by cryo-electron microscopy. The 135S and 80S particles are both ∼4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 Å were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell. PMID:10627545

  11. Antiviral Activity of Sulfated Polysaccharide of Adenanthera pavonina against Poliovirus in HEp-2 Cells

    PubMed Central

    de Godoi, Ananda Marques; Faccin-Galhardi, Lígia Carla; Lopes, Nayara; de Almeida, Raimundo Rafael; Ricardo, Nágila Maria Pontes Silva; Nozawa, Carlos; Linhares, Rosa Elisa Carvalho

    2014-01-01

    Adenanthera pavonina, popularly known as red-bead tree, carolina, pigeon's eye, and dragon's eye, is a plant traditionally used in Brazil for the treatment of several diseases. The present study aimed at evaluating the activity of sulfated polysaccharide from the Adenanthera pavonina (SPLSAp) seeds against poliovirus type 1 (PV-1) in HEp-2 cell cultures. The SPLSAp presented a cytotoxic concentration (CC50) of 500 μg/mL in HEp-2 cell cultures, evaluated by the dimethylthiazolyl-diphenyltetrazolium bromide method (MTT). The SPLSAp exhibited a significant antiviral activity, with a 50% inhibitory concentration (IC50) of 1.18 µg/mL, determined by plaque reduction assay and a high selectivity index (SI) of 423. The maximum inhibition (100%) of PV replication was found when the SPLSAp treatment was concomitant with viral infection (time 0 h), at all tested concentrations. The maximal inhibition was also found when the SPLSAp was used 1 h and 2 h postinfection, albeit at 50 μg/mL and 100 μg/mL. Therefore, we demonstrated that the SPLSAp inhibited PV growth. We also suggested that SPLSAp inhibited PV in more than one step of the replication, as the mechanism of antiviral action. We, therefore, selected the compound as a potential candidate for further development towards the control of the infection. PMID:25221609

  12. 5′ and 3′ Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves

    PubMed Central

    Diamos, Andrew G.; Rosenthal, Sun H.; Mason, Hugh S.

    2016-01-01

    We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5′ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5′ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5′ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody

  13. Infected dendritic cells are sufficient to mediate the adjuvant activity generated by Venezuelan equine encephalitis virus replicon particles

    PubMed Central

    Tonkin, Daniel R; Whitmore, Alan; Johnston, Robert E; Barro, Mario

    2012-01-01

    Replicon particles derived from Venezuelan equine encephalitis virus (VEE) are infectious non-propagating particles which act as a safe and potent systemic, mucosal, and cellular adjuvant when delivered with antigen. VEE and VEE replicon particles (VRP) can target multiple cell types including dendritic cells (DCs). The role of these cell types in VRP adjuvant activity has not been previously evaluated, and for these studies we focused on the contribution of DCs to the response to VRP. By analysis of VRP targeting in the draining lymph node, we found that VRP induced rapid recruitment of TNF-secreting monocyte-derived inflammatory dendritic cells. VRP preferentially infected these inflammatory DCs as well as classical DCs and macrophages, with less efficient infection of other cell types. DC depletion suggested that the interaction of VRP with classical DCs was required for recruitment of inflammatory DCs, induction of high levels of many cytokines, and for stable transport of VRP to the draining lymph node. Additionally, in vitro-infected DCs enhanced antigen-specific responses by CD4 and CD8 T cells. By transfer of VRP-infected DCs into mice we showed that these DCs generated an inflammatory state in the draining lymph node similar to that achieved by VRP injection. Most importantly, VRP-infected DCs were sufficient to establish robust adjuvant activity in mice comparable to that produced by VRP injection. These findings indicate that VRP infect, recruit and activate both classical and inflammatory DCs, and those DCs become mediators of the VRP adjuvant activity. PMID:22531556

  14. Vaccination with Recombinant RNA Replicon Particles Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

    PubMed Central

    Halbherr, Stefan J.; Brostoff, Terza; Tippenhauer, Merve; Locher, Samira; Berger Rentsch, Marianne; Zimmer, Gert

    2013-01-01

    Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×108 infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry. PMID:23762463

  15. Molecular Smallpox Vaccine Delivered by Alphavirus Replicons Elicits Protective Immunity in Mice and Non-human Primates

    PubMed Central

    Hooper, Jay W.; Ferro, Anthony M.; Golden, Joseph W.; Silvera, Peter; Dudek, Jeanne; Alterson, Kim; Custer, Max; Rivers, Bryan; Morris, John; Owens, Gary; Smith, Jonathan F.; Kamrud, Kurt I.

    2009-01-01

    Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 70s. Because of its highly contagious nature and high mortality rate, smallpox has significant potential as a biological weapon. Unfortunately, the current vaccine for orthopoxviruses is contraindicated for large portions of the population. Thus, there is a need for new, safe, and effective orthopoxvirus vaccines. Alphavirus replicon vectors, derived from strains of Venezuelan equine encephalitis virus, are being used to develop alternatives to the current smallpox vaccine. Here, we demonstrated that virus-like replicon particles (VRP) expressing the vaccinia virus A33R, B5R, A27L, and L1R genes elicited protective immunity in mice comparable to vaccination with live-vaccinia virus. Furthermore, cynomolgus macaques vaccinated with a combination of the four poxvirus VRPs (4pox-VRP) developed antibody responses to each antigen. These antibody responses were able to neutralize and inhibit the spread of both vaccinia virus and monkeypox virus. Macaques vaccinated with 4pox-VRP, flu HA VRP (negative control), or live-vaccinia virus (positive control) were challenged intravenously with 5 × 106 PFU of monkeypox virus 1 month after the second VRP vaccination. Four of the six negative control animals succumbed to monkeypox and the remaining two animals demonstrated either severe or grave disease. Importantly, all 10 macaques vaccinated with the 4pox-VRP vaccine survived without developing severe disease. These findings revealed that a single-boost VRP smallpox vaccine shows promise as a safe alternative to the currently licensed live-vaccinia virus smallpox vaccine. PMID:19833247

  16. Burkholderia xernovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility

    SciTech Connect

    Chain, Patrick S. G.; Denef, Vincent; Konstantinidis, Konstantinos T; Vergez, Lisa; Agullo, Loreine; Reyes, Valeria Latorre; Hauser, Loren John; Cordova, Macarena; Gomez, Luis; Gonzalez, Myriam; Land, Miriam L; Lao, Victoria; Larimer, Frank W; LiPuma, John J; Mahenthiralingam, Eshwar; Malfatti, Stephanie; Marx, Christopher J; Parnell, J Jacob; Ramette, Alban; Richardson, P M; Seeger, Michael; Smith, Daryl; Spilker, Theodore; Sul, Woo Jun; Tsoi, Tamara V; Zhulin, Igor B; Tiedje, James M.

    2006-01-01

    Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven 'central aromatic' and twenty 'peripheral aromatic' pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.

  17. The Dual Role of an ESCRT-0 Component HGS in HBV Transcription and Naked Capsid Secretion

    PubMed Central

    Chou, Shu-Fan; Tsai, Ming-Lin; Huang, Jyun-Yuan; Chang, Ya-Shu; Shih, Chiaho

    2015-01-01

    The Endosomal Sorting Complex Required for Transport (ESCRT) is an important cellular machinery for the sorting and trafficking of ubiquitinated cargos. It is also known that ESCRT is required for the egress of a number of viruses. To investigate the relationship between ESCRT and hepatitis B virus (HBV), we conducted an siRNA screening of ESCRT components for their potential effect on HBV replication and virion release. We identified a number of ESCRT factors required for HBV replication, and focused our study here on HGS (HRS, hepatocyte growth factor-regulated tyrosine kinase substrate) in the ESCRT-0 complex. Aberrant levels of HGS suppressed HBV transcription, replication and virion secretion. Hydrodynamic delivery of HGS in a mouse model significantly suppressed viral replication in the liver and virion secretion in the serum. Surprisingly, overexpression of HGS stimulated the release of HBV naked capsids, irrespective of their viral RNA, DNA, or empty contents. Mutant core protein (HBc 1–147) containing no arginine-rich domain (ARD) failed to secrete empty virions with or without HGS. In contrast, empty naked capsids of HBc 1–147 could still be promoted for secretion by HGS. HGS exerted a strong positive effect on the secretion of naked capsids, at the expense of a reduced level of virions. The association between HGS and HBc appears to be ubiquitin-independent. Furthermore, HBc is preferentially co-localized with HGS near the cell periphery, instead of near the punctate endosomes in the cytoplasm. In summary, our work demonstrated the importance of an optimum level of HGS in HBV propagation. In addition to an effect on HBV transcription, HGS can diminish the pool size of intracellular nucleocapsids with ongoing genome maturation, probably in part by promoting the secretion of naked capsids. The secretion routes of HBV virions and naked capsids can be clearly distinguished based on the pleiotropic effect of HGS involved in the ESCRT-0 complex. PMID

  18. Poliovirus protease 3C (P3-7c) does not cleave P220 of the eucaryotic mRNA cap-binding protein complex.

    PubMed Central

    Lee, K A; Edery, I; Hanecak, R; Wimmer, E; Sonenberg, N

    1985-01-01

    Infection of HeLa cells by poliovirus results in proteolysis of the large subunit (P220) of the cap-binding protein complex. This is believed to cause the rapid shut-off of host protein synthesis during poliovirus infection. In this communication we examined the possible involvement of poliovirus proteins 3C (a proteinase) and 2C in cleavage of P220. Using antisera against these two viral polypeptides, we were unable to inhibit proteolysis of P220 in an in vitro assay. These results indicate that viral proteins 3C and 2C are not directly involved in cleaving P220 and hence do not cause shut-off of cellular protein synthesis. Images PMID:2991572

  19. Marked increases in concentrations of apolipoprotein in the cerebrospinal fluid of poliovirus-infected macaques: relations between apolipoprotein concentrations and severity of brain injury.

    PubMed Central

    Saito, K; Seishima, M; Heyes, M P; Song, H; Fujigaki, S; Maeda, S; Vickers, J H; Noma, A

    1997-01-01

    Apolipoproteins in cerebrospinal fluid (CSF) might have important functional roles in the pathophysiology of brain and lipid metabolism in the vascular component. The present study examined apolipoprotein A-I (apo-A-I) and apolipoprotein E (apo-E) levels in CSF and serum from poliovirus-infected macaques. Poliovirus-infected macaques developed motor deficits and were classified into three groups: (1) muscle weakness in one or both legs; (2) partial paralysis in one or both legs; (3) complete paralysis in one or both legs. No motor deficits were evident in the control or sham-treated macaques. Apo-A-I concentrations in CSF were markedly elevated in poliovirus-infected macaques with weakness, partial or complete paralysis, in comparison with either control or sham-treated animals, and were proportional to the severity of motor impairment. Apo-E concentrations in CSF were also significantly elevated in poliovirus-infected macaques with complete paralysis. The magnitude of increase in CSF apo-A-I or apo-E concentrations was also closely associated with the degree of histologic neurological damage and inflammation (lesion scores). However, no changes in serum apo-A-I and apo-E concentrations were observed in the poliovirus-infected macaques compared with control macaques. Furthermore there were no significant correlations apo-A-I or apo-E concentrations between serum and CSF. We hypothesize that the elevation of apo-A-I and apo-E concentrations after poliovirus infection is caused by immune stimulation within the central nervous system (CNS). Measures of CSF apo-A-I and apo-E levels might serve as a useful marker for the severity and/or the range of CNS injury. PMID:9003413

  20. Inhibition of endoplasmic reticulum-to-Golgi traffic by poliovirus protein 3A: genetic and ultrastructural analysis.

    PubMed Central

    Doedens, J R; Giddings, T H; Kirkegaard, K

    1997-01-01

    Poliovirus protein 3A, only 87 amino acids in length, is a potent inhibitor of protein secretion in mammalian cells, blocking anterograde protein traffic from the endoplasmic reticulum (ER) to the Golgi complex. The function of viral protein 3A in blocking protein secretion is extremely sensitive to mutations near the N terminus of the protein. Deletion of the first 10 amino acids or insertion of a single amino acid between amino acids 15 and 16, a mutation that causes a cold-sensitive defect in poliovirus RNA replication, abrogates the inhibition of protein secretion although wild-type amounts of the mutant proteins are expressed. Immunofluorescence light microscopy and immunoelectron microscopy demonstrate that 3A protein, expressed in the absence of other viral proteins, colocalizes with membranes derived from the ER. The precise topology of 3A with respect to ER membranes is not known, but it is likely to be associated with the cytosolic surface of the ER. Although the glycosylation of 3A in translation extracts has been reported, we show that tunicamycin, under conditions in which glycosylation of cellular proteins is inhibited, has no effect on poliovirus growth. Therefore, glycosylation of 3A plays no functional role in the viral replicative cycle. Electron microscopy reveals that the ER dilates dramatically in the presence of 3A protein. The absence of accumulated vesicles and the swelling of the ER-derived membranes argues that ER-to-Golgi traffic is inhibited at the step of vesicle formation or budding from the ER. PMID:9371562

  1. Degradation of the interferon-induced 68,000-M(r) protein kinase by poliovirus requires RNA.

    PubMed Central

    Black, T L; Barber, G N; Katze, M G

    1993-01-01

    Control of the interferon-induced double-stranded RNA (dsRNA) activated protein kinase (referred to as P68 because of its M(r) of 68,000 in human cells) by animal viruses is essential to avoid decreases in protein synthetic rates during infection. We have previously demonstrated that poliovirus establishes a unique way of regulating the protein kinase, namely by inducing the specific degradation of P68 during infection (T. L. Black, B. Safer, A. Hovanessian, and M. G. Katze, J. Virol. 63:2244-2251, 1989). In the present study we investigated the mechanisms by which P68 degradation occurred. To do this we used an in vitro degradation assay which faithfully reproduced the in vivo events. Although viral gene expression was required for P68 degradation, the major poliovirus proteases, 2A and 3C, were found not to be directly involved with P68 proteolysis. However, the protease responsible for P68 degradation required divalent cations for maximal activity and probably has both an RNA and a protein component since trypsin and ribonuclease abrogated the activity. Despite this requirement for divalent cations and RNA, activation of the kinase was not required for proteolysis since a catalytically inactive P68 was still degraded. Mapping of P68 protease-sensitive sites by using in vitro translated truncation and deletion mutants revealed that sites required for degradation resided in the amino terminus and colocalized to dsRNA-binding domains. Finally, we found that preincubation of cell extracts with the synthetic dsRNA poly(I-C) largely prevented P68 proteolysis, providing additional evidence for the critical role of RNA. On the basis of these data, we present a hypothetical model depicting possible mechanisms of P68 degradation in poliovirus-infected cells. Images PMID:7678306

  2. Influence of Enteric Infections on Response to Oral Poliovirus Vaccine: A Systematic Review and Meta-analysis

    PubMed Central

    Parker, Edward P. K.; Kampmann, Beate; Kang, Gagandeep; Grassly, Nicholas C.

    2014-01-01

    Background. The impaired immunogenicity of oral poliovirus vaccine (OPV) in low-income countries has been apparent since the early field trials of this vaccine. Infection with enteropathogens at the time of vaccination may contribute to this phenomenon. However, the relative influence of these infections on OPV performance remains uncertain. Methods. We conducted a systematic review to examine the impact of concurrent enteric infections on OPV response. Using random-effects models, we assessed the effects of nonpolio enteroviruses (NPEVs) and diarrhea on the odds of seroconversion and/or vaccine virus shedding. Results. We identified 25 trials in which OPV outcomes were compared according to the presence or absence of enteric infections, the majority of which (n = 17) reported only on NPEVs. Concurrent NPEVs significantly reduced the odds of per-dose seroconversion for type 1 poliovirus (odds ratio [OR] 0.44, 95% confidence interval 0.23−0.84), but not type 2 (OR 0.53 [0.19−1.46]) or type 3 (OR 0.56 [0.27−1.12]). A similar reduction, significant for type 1 poliovirus (OR 0.50 [0.28−0.89]), was observed in the odds of vaccine virus shedding among NPEV-infected individuals. Concurrent diarrhea significantly inhibited per-dose seroconversion overall (OR 0.61 [0.38−0.87]). Conclusions. Our findings are consistent with an inhibitory effect of concurrent enteric infections on OPV response. PMID:24688069

  3. Stabilization of Poliovirus Polymerase by NTP Binding and Fingers-Thumb Interactions

    PubMed Central

    Thompson, Aaron A.; Albertini, Rebecca A.; Peersen, Olve B.

    2007-01-01

    The viral RNA-dependent RNA polymerases show a conserved structure where the fingers domain interacts with the top of the thumb domain to create a tunnel through which nucleotide triphosphates reach the active site. We have solved the crystal structures of poliovirus polymerase (3Dpol) in complex with all four NTPs, showing that they all bind in a common preinsertion site where the phosphates are not yet positioned over the active site. The NTPs interact with both the fingers and palm domains, forming bridging interactions that explain the increased thermal stability of 3Dpol in the presence of NTPs. We have also examined the importance of the fingers-thumb domain interaction for the function and structural stability of 3Dpol. Results from thermal denaturation experiments using circular dichroism and 2-aniliino-6-napthaline-sulfonate (ANS) fluorescence show that 3Dpol has a melting temperature of only ∼40°C. NTP binding stabilizes the protein and increases the melting by 5-6 °C while mutations in the fingers-thumb domain interface destabilize the protein and reduce the melting point by as much as 6 °C. In particular, the burial of Phe30 and Phe34 from the tip of the index finger into a pocket at the top of the thumb and the presence of Trp403 on the thumb domain are key interactions required to maintain the structural integrity of the polymerase. The data suggest the fingers domain has significant conformational flexibility and exists in a highly dynamic molten globule state at physiological temperature. The role of the enclosed active site motif as a structural scaffold for constraining the fingers domain and accommodating conformational changes in 3Dpol and other viral polymerases during the catalytic cycle is discussed. PMID:17223130

  4. Influence of pH and electrolyte composition on adsorption of poliovirus by soils and minerals.

    PubMed Central

    Taylor, D H; Moore, R S; Sturman, L S

    1981-01-01

    The pH and the nature an concentration of simple electrolytes influenced the interaction of poliovirus type 2 with three soils, a sand, and a clay mineral. In electrolytes above pH 9 the virus was not adsorbed extensively to the substrates, but below pH 7 almost all virus was bound. For each adsorbent there was a characteristic pH region of transition from strong to weak uptake. Differences between the soils in virus uptake were shown to parallel their pH-dependent mineral. In electrolytes above pH 9 the virus was not adsorbed extensively to the substrates, but below pH 7 almost all virus was bound. For each adsorbent there was a characteristic pH region of transition from strong to weak uptake. Differences between the soils in virus uptake were shown to parallel their pH-dependent mineral. In electrolytes above pH 9 the virus was not adsorbed extensively to the substrates, but below pH 7 almost all virus was bound. For each adsorbent there was a characteristic pH region of transition from strong to weak uptake. Differences between the soils in virus uptake were shown to parallel their pH-dependent charge properties, as determined by whole-particle microelectrophoresis. Only when the pH was close to or above the critical region was uptake increased with electrolyte concentration. The transition region for all substrates was above pH 7.5 the isoelectric point of the virus. Thus, it appears that when both the virus and substrate are highly negative charged, repulsive electrostatic effects may exceed inherent attractive interactions, thereby inhibiting adsorption. PMID:6274260

  5. [Genetic Characteristics of Type 2 Vaccine-derived Poliovirus in Shanxi Province (China) in 2014].

    PubMed

    Yan, Dongrei; Li, Xiaolei; Zhang, Yong; Yang, Jianfang; Zhu, Shuangli; Wang, Dongyan; Zhang, Chuangye; Zhu, Hui; Xu, Wenbo

    2015-03-01

    The World Health Organization redefined the type 2 vaccine-derived poliovirus (VDPV) in 2010. To study the genetic characteristics and evolution of type 2 VDPV under this new definition, we conducted genome sequencing and analyses of type 2 VDPVs isolated from one patient with acute flaccid paralysis in Shanxi province (China) in 2014. Nucleotide sequencing revealed that the full-length of type 2 VDPV is 7439 bases encoding 2207 amino acids with no insertion or deletion of nucleotides compared with Sabin2. One nucleotide substitution identified as a key determinant of the attenuated phenotype of the Sabin 2 strain (A-G reversion at nucleotide nt 481 in the 5-end of the untranslated region) had reverted in the Shanxi type 2 VDPV. The other known key determinant of the attenuated phenotype of the Sabin 2 strain (U-->C reversion at nt2909 in the VP1 coding region that caused a Ile143Thr substitution in VP1) had not reverted in the Shanxi VDPV. The Shanxi type 2 VDPV was S2/S1 recombinant, the crossover site of which mapped to the 3-end of the 3D region (between nt 6247 and nt 6281). A phylogentic tree based on the VP1 coding region showed that evolution of the Shanxi type 2 VDPV was independent of other type 2 VDPVs detected worldwide. We estimated that the strain circulated for approximately = 11 months in the population according to the known evolution rate. The present study confirmed that the Chinese Polio Laboratory Network could discover the VDPV promptly and that it played an important part in maintenance of a polio-free China. PMID:26164941

  6. The recent outbreaks and reemergence of poliovirus in war and conflict-affected areas

    PubMed Central

    Akil, Luma; Ahmad, H. Anwar

    2016-01-01

    SUMMARY Background Poliomyelitis is a highly infectious disease caused by poliovirus, which becomes difficult to manage/eradicate in politically unstable areas. The objectives of this study were to determine the movement and management of such polio outbreaks in endemic countries and countries with reoccurring cases of polio and to determine the effect of political instability on polio eradication. Methods In this study, the extent of polio outbreaks was examined and modeled using statistical methodologies and mapped with GIS software. Data on polio cases and immunization were collected for countries with polio cases for the period 2011 to 2014. Weekly data from the Global Polio Eradication Initiative were collected for selected countries. The recent virus origin and current movement was mapped using GIS. Correlations between immunization rates, the Global Peace Index (GPI), and other indicators of a country’s political stability with polio outbreaks were determined. Data were analyzed using SAS 9.4 and ArcGIS 10. Results For several reasons, Pakistan remains highly vulnerable to new incidences of polio (306 cases in 2014). Overall immunization rates showed a steady decline over time in selected countries. Countries with polio cases were shown to have high rates of infant mortality, and their GPI ranked between 2.0 and 3.3; displaced populations, level of violent crime rating, and political instability also were ranked high for several countries. Conclusion Polio was shown to be high in areas with increased conflict and instability. Displaced populations living in hard-to-reach areas may lack access to proper vaccination and health care. Wars and conflict have also resulted in the reemergence of polio in otherwise polio-free countries. PMID:27237735

  7. Transferrin Receptor 1 Facilitates Poliovirus Permeation of Mouse Brain Capillary Endothelial Cells.

    PubMed

    Mizutani, Taketoshi; Ishizaka, Aya; Nihei, Coh-Ichi

    2016-02-01

    As a possible route for invasion of the CNS, circulating poliovirus (PV) in the blood is believed to traverse the blood-brain barrier (BBB), resulting in paralytic poliomyelitis. However, the underlying mechanism is poorly understood. In this study, we demonstrated that mouse transferrin receptor 1 (mTfR1) is responsible for PV attachment to the cell surface, allowing invasion into the CNS via the BBB. PV interacts with the apical domain of mTfR1 on mouse brain capillary endothelial cells (MBEC4) in a dose-dependent manner via its capsid protein (VP1). We found that F-G, G-H, and H-I loops in VP1 are important for this binding. However, C-D, D-E, and E-F loops in VP1-fused Venus proteins efficiently penetrate MBEC4 cells. These results imply that the VP1 functional domain responsible for cell attachment is different from that involved in viral permeation of the brain capillary endothelium. We observed that co-treatment of MBEC4 cells with excess PV particles but not dextran resulted in blockage of transferrin transport into cells. Using the Transwell in vitro BBB model, transferrin co-treatment inhibited permeation of PV into MBEC4 cells and delayed further viral permeation via mTfR1 knockdown. With mTfR1 as a positive mediator of PV-host cell attachment and PV permeation of MBEC4 cells, our results indicate a novel role of TfR1 as a cellular receptor for human PV receptor/CD155-independent PV invasion of the CNS. PMID:26637351

  8. Antiviral effect of Guazuma ulmifolia and Stryphnodendron adstringens on poliovirus and bovine herpesvirus.

    PubMed

    Felipe, Adriana Meri Mestrimer; Rincão, Vinicius Pires; Benati, Fabrício José; Linhares, Rosa Elisa Carvalho; Galina, Karen Janaina; de Toledo, Cleyton Eduardo Mendes; Lopes, Gisely Cristiny; de Mello, João Carlos Palazzo; Nozawa, Carlos

    2006-06-01

    Crude extract (CE) and aqueous (AqF) and ethyl acetate (EtOAcF) fractions of Guazuma ulmifolia Lam., Sterculiaceae and the corresponding AqF, EtOAcF of Stryphnodendron adstringens (Mart.) Coville, Leguminosae were tested for their antiviral activity against poliovirus 1 (P-1) and bovine herpesvirus 1 (BHV-1) in HEp-2 cultured cells. The antiviral activity was monitored by plaque assay and immunofluorescence assay (IFA) under virucidal and therapeutic protocols. The therapeutic protocol demonstrated statistically significant positive results with both plants and for both virus strains. The highest percentages of viral inhibition were found for G. ulmifolia EtOAcF which inhibited BHV-1 and P-1 replication by 100% and 99%, respectively (p<0.05, Student's t-test). For S. adstringens, AqF was the most efficient, inhibiting BHV-1 and P-1 by 97% and 93%, respectively (p<0.05). In the virucidal protocol, G. ulmifolia CE inhibited the replication of BHV-1 and P-1 by 60% and 26%, respectively (p<0.05), while, for S. adstringens, inhibition of 62% (p<0.05) was demonstrated only with EtOAcF for P-1. IFA demonstrated that the greatest reduction in fluorescent cell number occurred with G. ulmifolia, under the therapeutic protocol for both virus strains. However, AqF and EtOAcF of S. adstringens were most efficient with the virucidal protocol for P-1. In conclusion, we demonstrated that G. ulmifolia and S. adstringens inhibited BHV-1 and P-1 replication, as well as, blocked the synthesis of viral antigens in infected cell cultures. PMID:16754999

  9. Electromagnetic radiation due to naked singularity formation in self-similar gravitational collapse

    SciTech Connect

    Mitsuda, Eiji; Yoshino, Hirotaka; Tomimatsu, Akira

    2005-04-15

    Dynamical evolution of test fields in background geometry with a naked singularity is an important problem relevant to the Cauchy horizon instability and the observational signatures different from black hole formation. In this paper we study electromagnetic perturbations generated by a given current distribution in collapsing matter under a spherically symmetric self-similar background. Using the Green's function method, we construct the formula to evaluate the outgoing energy flux observed at the future null infinity. The contributions from 'quasinormal' modes of the self-similar system as well as 'high-frequency' waves are clarified. We find a characteristic power-law time evolution of the outgoing energy flux which appears just before naked singularity formation and give the criteria as to whether or not the outgoing energy flux diverges at the future Cauchy horizon.

  10. Asymptotic behavior of dynamical variables and naked singularity formation in spherically symmetric gravitational collapse

    SciTech Connect

    Kawakami, Hayato; Mitsuda, Eiji; Nambu, Yasusada; Tomimatsu, Akira

    2009-07-15

    In considering the gravitational collapse of matter, it is an important problem to clarify what kind of conditions leads to the formation of naked singularity. For this purpose, we apply the 1+3 orthonormal frame formalism introduced by Uggla et al. to the spherically symmetric gravitational collapse of a perfect fluid. This formalism allows us to construct an autonomous system of evolution and constraint equations for scale-invariant dynamical variables normalized by the volume expansion rate of the timelike orthonormal frame vector. We investigate the asymptotic evolution of such dynamical variables towards the formation of a central singularity and present a conjecture that the steep spatial gradient for the normalized density function is a characteristic of the naked singularity formation.

  11. A naked eye refractive index sensor with a visible multiple peak metamaterial absorber.

    PubMed

    Ma, Heli; Song, Kun; Zhou, Liang; Zhao, Xiaopeng

    2015-01-01

    We report a naked eye refractive index sensor with a visible metamaterial absorber. The visible metamaterial absorber consisting of a silver dendritic/dielectric/metal structure shows multiple absorption peaks. By incorporating a gain material (rhodamine B) into the dielectric layer, the maximal magnitude of the absorption peak can be improved by about 30%. As the metamaterial absorber is sensitive to the refractive index of glucose solutions, it can function as a sensor that quickly responds to variations of the refractive index of the liquid. Meanwhile, since the response is presented via color changes, it can be clearly observed by the naked eyes. Further experiments have confirmed that the sensor can be used repeatedly. PMID:25822141

  12. Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye

    NASA Astrophysics Data System (ADS)

    de La Rica, Roberto; Stevens, Molly M.

    2012-12-01

    In resource-constrained countries, affordable methodologies for the detection of disease biomarkers at ultralow concentrations can potentially improve the standard of living. However, current strategies for ultrasensitive detection often require sophisticated instruments that may not be available in laboratories with fewer resources. Here, we circumvent this problem by introducing a signal generation mechanism for biosensing that enables the detection of a few molecules of analyte with the naked eye. The enzyme label of an enzyme-linked immunosorbent assay (ELISA) controls the growth of gold nanoparticles and generates coloured solutions with distinct tonality when the analyte is present. Prostate specific antigen (PSA) and HIV-1 capsid antigen p24 were detected in whole serum at the ultralow concentration of 1 × 10-18 g ml-1. p24 was also detected with the naked eye in the sera of HIV-infected patients showing viral loads undetectable by a gold standard nucleic acid-based test.

  13. Occurrence of 20S RNA and 23S RNA replicons in industrial yeast strains and their variation under nutritional stress conditions.

    PubMed

    López, Victoria; Gil, Rosario; Vicente Carbonell, José; Navarro, Alfonso

    2002-04-01

    We have characterized industrial yeast strains used in the brewing, baking, and winemaking industries for the presence or absence of cytoplasmic single-stranded 20S and 23S RNAs. Furthermore, the variation of intracellular concentrations of these replicons in brewing and laboratory strains under nutritional stress conditions was determined. Our results show a correlation between the relative abundance of these replicons and exposure of yeast to nutritionally stressful conditions, indicating that these RNAs could be employed as molecular probes to evaluate the exposure of 20S(+) and/or 23S(+) yeast strains to stress situations during industrial manipulation. During this study, several 20S(-)23S(+) Saccharomyces cerevisiae strains were isolated and identified. This is the first time that a yeast strain containing only 23S RNA has been reported, demonstrating that 20S RNA is not required for 23S RNA replication. PMID:11921103

  14. Unraveling the message: insights into comparative genomics of the naked mole-rat.

    PubMed

    Lewis, Kaitlyn N; Soifer, Ilya; Melamud, Eugene; Roy, Margaret; McIsaac, R Scott; Hibbs, Matthew; Buffenstein, Rochelle

    2016-08-01

    Animals have evolved to survive, and even thrive, in different environments. Genetic adaptations may have indirectly created phenotypes that also resulted in a longer lifespan. One example of this phenomenon is the preternaturally long-lived naked mole-rat. This strictly subterranean rodent tolerates hypoxia, hypercapnia, and soil-based toxins. Naked mole-rats also exhibit pronounced resistance to cancer and an attenuated decline of many physiological characteristics that often decline as mammals age. Elucidating mechanisms that give rise to their unique phenotypes will lead to better understanding of subterranean ecophysiology and biology of aging. Comparative genomics could be a useful tool in this regard. Since the publication of a naked mole-rat genome assembly in 2011, analyses of genomic and transcriptomic data have enabled a clearer understanding of mole-rat evolutionary history and suggested molecular pathways (e.g., NRF2-signaling activation and DNA damage repair mechanisms) that may explain the extraordinarily longevity and unique health traits of this species. However, careful scrutiny and re-analysis suggest that some identified features result from incorrect or imprecise annotation and assembly of the naked mole-rat genome: in addition, some of these conclusions (e.g., genes involved in cancer resistance and hairlessness) are rejected when the analysis includes additional, more closely related species. We describe how the combination of better study design, improved genomic sequencing techniques, and new bioinformatic and data analytical tools will improve comparative genomics and ultimately bridge the gap between traditional model and nonmodel organisms. PMID:27364349

  15. String loops in the field of braneworld spherically symmetric black holes and naked singularities

    SciTech Connect

    Stuchlík, Z.; Kološ, M. E-mail: martin.kolos@fpf.slu.cz

    2012-10-01

    We study motion of current-carrying string loops in the field of braneworld spherically symmetric black holes and naked singularities. The spacetime is described by the Reissner-Nordström geometry with tidal charge b reflecting the non-local tidal effects coming from the external dimension; both positive and negative values of the spacetime parameter b are considered. We restrict attention to the axisymmetric motion of string loops when the motion can be fully governed by an appropriately defined effective potential related to the energy and angular momentum of the string loops. In dependence on these two constants of the motion, the string loops can be captured, trapped, or can escape to infinity. In close vicinity of stable equilibrium points at the centre of trapped states the motion is regular. We describe how it is transformed to chaotic motion with growing energy of the string loop. In the field of naked singularities the trapped states located off the equatorial plane of the system exist and trajectories unable to cross the equatorial plane occur, contrary to the trajectories in the field of black holes where crossing the equatorial plane is always admitted. We concentrate our attention to the so called transmutation effect when the string loops are accelerated in the deep gravitational field near the black hole or naked singularity by transforming the oscillatory energy to the energy of the transitional motion. We demonstrate that the influence of the tidal charge can be substantial especially in the naked singularity spacetimes with b > 1 where the acceleration to ultrarelativistic velocities with Lorentz factor γ ∼ 100 can be reached, being more than one order higher in comparison with those obtained in the black hole spacetimes.

  16. Digital dissection of the masticatory muscles of the naked mole-rat, Heterocephalus glaber (Mammalia, Rodentia)

    PubMed Central

    Faulkes, Chris G.

    2014-01-01

    The naked mole-rat, Heterocephalus glaber, of the family Bathyergidae is a subterranean rodent that feeds on underground roots and tubers and digs extensive tunnel systems with its incisors. It is a highly unusual mammal with regard to its social structure, longevity, pain insensitivity and cancer resistance, all of which have made it the subject of a great deal of research in recent years. Yet, much of the basic anatomy of this species remains undocumented. In this paper, we describe the morphology of the jaw-closing musculature of the naked mole-rat, as revealed by contrast-enhanced micro-computed tomography. This technique uses an iodine stain to enable the imaging of soft tissues with microCT. The iodine-enhanced scans were used to create 3D reconstructions of the naked mole-rat masticatory muscles from which muscle masses were calculated. The jaw-closing musculature of Heterocephalus glaber is relatively very large compared to other rodents and is dominated by the superficial masseter, the deep masseter and the temporalis. The temporalis in particular is large for a rodent, covering the entirety of the braincase and much of the rear part of the orbit. The morphology of the masseter complex described here differs from two other published descriptions of bathyergid masticatory muscles, but is more similar to the arrangement seen in other rodent families. The zygomaticomandibularis (ZM) muscle does not protrude through the infraorbital foramen on to the rostrum and thus the naked mole-rat should be considered protrogomorphous rather than hystricomorphous, and the morphology is consistent with secondarily lost hystricomorphy as has been previously suggested for Bathyergidae. Overall, the morphology of the masticatory musculature indicates a species with a high bite force and a wide gape–both important adaptations for a life dominated by digging with the incisors. PMID:25024917

  17. A Cyanine Dye Encapsulated Porous Fibrous Mat for Naked-Eye Ammonia Sensing.

    PubMed

    Ji, Chendong; Ma, Lijing; Yin, Meizhen; Yang, Wantai; Pan, Kai

    2016-08-19

    Electrospun ultrathin fiber-based sensors are desirable because of their practicality and sensitivity. Ammonia-detection systems are in high demand in different areas, including the industrial and agricultural fields. However, current technologies rely on large and complex instruments that restrict their actual utilization. Herein, we report a flexible naked-eye ammonia sensor, the polylactic acid-cyanine (PLA-Cy) fibrous mat, which was fabricated by blending a carboxyl-functionalized cyanine dye (D1) into electospun PLA porous fibers. The sensing mat was shown to undergo a naked-eye-detectable color change from white to blue upon exposure to ammonia vapor. The mat showed high selectivity to ammonia gas with a detection limit of 3.3 ppm. Aggregated D1 was first encapsulated by PLA and was then ionized by NH3 . These mechanisms were examined by photophysical studies and scanning electron microscopy. The aggregation-deaggregation process of D1 in the PLA-Cy fibrous mat led to the color change. This work provides a facile method for the naked-eye detection of ammonia and a novel strategy for the use of organic dyes in ammonia sensing. PMID:27411059

  18. Gis-Based Crop Support System For Common Oatand Naked Oat in China

    NASA Astrophysics Data System (ADS)

    Wan, Fan; Wang, Zhen; Li, Fengmin; Cao, Huhua; Sun, Guojun

    The identification of the suitable areas for common oat (Avena sativa L.) and naked oat (Avena nuda L.) in China using Multi-Criteria Evaluation (MCE) approach based on GIS is presented in the current article. Climate, topography, soil, land use and oat variety databases were created. Relevant criteria,suitability levels and their weights for each factor were defined. Then the criteria maps were obtained and turned into the MCE process, and suitability maps for common oat and naked oat were created. The land use and the suitability maps were crossed to identify the suitable areas for each crop. The results identified 397,720 km2 of suitable areas for common oats of forage purpose distributed in 744 counties in 17 provinces, and 556,232 km2 of suitable areas for naked oats of grain purpose distributed in 779 counties in 19 provinces. This result is in accordance with the distribution of farmingpastoral ecozones located in semi-arid regions of northern China. The mapped areas can help define the working limits and serve as indicative zones for oat in China. The created databases, mapped results, interface of expert system and relevant hardware facilities could construct a complete crop support system for oats.

  19. Optical effects related to Keplerian discs orbiting Kehagias-Sfetsos naked singularities

    NASA Astrophysics Data System (ADS)

    Stuchlík, Zdeněk; Schee, Jan

    2014-10-01

    We demonstrate possible optical signatures of the Kehagias-Sfetsos (KS) naked singularity spacetimes representing a spherically symmetric vacuum solution of the modified Hořava gravity. In such spacetimes, accretion structures significantly different from those present in standard black hole spacetimes occur due to the ‘antigravity’ effect, which causes an internal static sphere surrounded by Keplerian discs. We focus our attention on the optical effects related to the Keplerian accretion discs, constructing the optical appearance of the Keplerian discs, the spectral continuum due to their thermal radiation, and the spectral profiled lines generated in the innermost parts of such discs. The KS naked singularity signature is strongly encoded in the characteristics of predicted optical effects, especially in cases where the spectral continuum and spectral lines are profiled by the strong gravity of the spacetimes due to the vanishing region of the angular velocity gradient influencing the effectiveness of the viscosity mechanism. We can conclude that optical signatures of KS naked singularities can be well distinguished from the signatures of standard black holes.

  20. Structural Features of the Telomerase RNA Gene in the Naked Mole Rat Heterocephalus glaber

    PubMed Central

    Evfratov, S. A.; Smekalova, E. M.; Golovin, A. V.; Logvina, N. A.; Zvereva, M. I.; Dontsova, O. A.

    2014-01-01

    Telomere length, an important feature of life span control, is dependent on the activity of telomerase (a key enzyme of the telomere-length-maintaining system). Telomerase RNA is a component of telomerase and, thus, is crucial for its activity. The structures of telomerase RNA genes and their promoter regions were compared for the long-living naked mole rat and different organisms. Two rare polymorphisms in Heterocephalus glaber telomerase RNA (hgTER) were identified: A→G in the first loop of pseudoknot P2b-p3 (an equivalent of 111nt in hTR) and G→A in the scaRNA domain CR7-p8b (an equivalent of 421nt in hTR). Analysis of TER promoter regions allowed us to identify two new transcription factor binding sites. The first one is the ETS family site, which was found to be a conserved element for all the analyzed TER promoters. The second site is unique for the promoter region of TER of the naked mole rat and is a binding site for the SOX17 transcription factor. The absence of one Sp1 site in the TER promoter region of the naked small rat is an additional specific feature of the promoter area of hgTER. Such variation in the hgTER transcription regulation region and hgTER itself could provide increased telomerase activity in stem cells and an extended lifespan to H. glaber. PMID:25093110

  1. A paper-based cantilever array sensor: Monitoring volatile organic compounds with naked eye.

    PubMed

    Fraiwan, Arwa; Lee, Hankeun; Choi, Seokheun

    2016-09-01

    Volatile organic compound (VOC) detection is critical for controlling industrial and commercial emissions, environmental monitoring, and public health. Simple, portable, rapid and low-cost VOC sensing platforms offer the benefits of on-site and real-time monitoring anytime and anywhere. The best and most practically useful approaches to monitoring would include equipment-free and power-free detection by the naked eye. In this work, we created a novel, paper-based cantilever sensor array that allows simple and rapid naked-eye VOC detection without the need for power, electronics or readout interface/equipment. This simple VOC detection method was achieved using (i) low-cost paper materials as a substrate and (ii) swellable thin polymers adhered to the paper. Upon exposure to VOCs, the polymer swelling adhered to the paper-based cantilever, inducing mechanical deflection that generated a distinctive composite pattern of the deflection angles for a specific VOC. The angle is directly measured by the naked eye on a 3-D protractor printed on a paper facing the cantilevers. The generated angle patterns are subjected to statistical algorithms (linear discriminant analysis (LDA)) to classify each VOC sample and selectively detect a VOC. We classified four VOC samples with 100% accuracy using LDA. PMID:27343578

  2. Ultrasensitive detection and rapid identification of multiple foodborne pathogens with the naked eyes.

    PubMed

    Zhang, Heng; Zhang, Yali; Lin, Yankui; Liang, Tongwen; Chen, Zhihua; Li, Jinfeng; Yue, Zhenfeng; Lv, Jingzhang; Jiang, Qing; Yi, Changqing

    2015-09-15

    In this study, a novel approach for ultrasensitive detection and rapid high-throughput identification of a panel of common foodborne pathogens with the naked eyes is presented. As a proof-of-concept application, a multiple pathogen analysis array is fabricated through immobilizing three specific polyT-capture probes which can respectively recognize rfbE gene (Escherichia coli O157:H7), invA gene (Salmonella enterica), inlA gene (Listeria monocytogenes) on the plastic substrates. PCR has been developed for amplification and labeling target genes of rfbE, invA, inlA with biotin. The biotinated target DNA is then captured onto the surface of plastic strips through specific DNA hybridization. The succeeding staining of biotinated DNA duplexes with avidin-horseradish peroxidise (AV-HRP) and biotinated anti-HRP antibody greatly amplifies the detectable signal through the multiple cycle signal amplification strategy, and thus realizing ultrasensitive and specific detection of the above three pathogens in food samples with the naked eyes. Results showed approximately 5 copies target pathogenic DNA could be detected with the naked eyes. This simple but very efficient colorimetric assay also show excellent anti-interference capability and good stability, and can be readily applied to point-of-care diagnosis. PMID:25909338

  3. Cutaneous and periodontal inputs to the cerebellum of the naked mole-rat (Heterocephalus glaber)

    PubMed Central

    Sarko, Diana K.; Leitch, Duncan B.; Catania, Kenneth C.

    2013-01-01

    The naked mole-rat (Heterocephalus glaber) is a small fossorial rodent with specialized dentition that is reflected by the large cortical area dedicated to representation of the prominent incisors. Due to naked mole-rats’ behavioral reliance on the incisors for digging and for manipulating objects, as well as their ability to move the lower incisors independently, we hypothesized that expanded somatosensory representations of the incisors would be present within the cerebellum in order to accommodate a greater degree of proprioceptive, cutaneous, and periodontal input. Multiunit electrophysiological recordings targeting the ansiform lobule were used to investigate tactile inputs from receptive fields on the entire body with a focus on the incisors. Similar to other rodents, a fractured somatotopy appeared to be present with discrete representations of the same receptive fields repeated within each folium of the cerebellum. These findings confirm the presence of somatosensory inputs to a large area of the naked mole-rat cerebellum with particularly extensive representations of the lower incisors and mystacial vibrissae. We speculate that these extensive inputs facilitate processing of tactile cues as part of a sensorimotor integration network that optimizes how sensory stimuli are acquired through active exploration and in turn adjusts motor outputs (such as independent movement of the lower incisors). These results highlight the diverse sensory specializations and corresponding brain organizational schemes that have evolved in different mammals to facilitate exploration of and interaction with their environment. PMID:24302898

  4. RNA-Seq Based Transcriptome Analysis of Hepatitis E Virus (HEV) and Hepatitis B Virus (HBV) Replicon Transfected Huh-7 Cells

    PubMed Central

    Thakral, Deepshi; Joshi, Prashant; Durgapal, Hemlata; Panda, Subrat Kumar

    2014-01-01

    Pathogenesis of hepatitis B virus (HBV) and hepatitis E virus (HEV) infection is as varied as they appear similar; while HBV causes an acute and/or chronic liver disease and hepatocellular carcinoma, HEV mostly causes an acute self-limiting disease. In both infections, host responses are crucial in disease establishment and/or virus clearance. In the wake of worsening prognosis described during HEV super-infection over chronic HBV hepatitis, we investigated the host responses by studying alterations in gene expression in liver cells (Huh-7 cell line) by transfection with HEV replicon only (HEV-only), HBV replicon only (HBV-only) and both HBV and HEV replicons (HBV+HEV). Virus replication was validated by strand-specific real-time RT-PCR for HEV and HBsAg ELISA of the culture supernatants for HBV. Indirect immunofluorescence for the respective viral proteins confirmed infection. Transcription profiling was carried out by RNA Sequencing (RNA-Seq) analysis of the poly-A enriched RNA from the transfected cells. Averages of 600 million bases within 5.6 million reads were sequenced in each sample and ∼15,800 genes were mapped with at least one or more reads. A total of 461 genes in HBV+HEV, 408 in HBV-only and 306 in HEV-only groups were differentially expressed as compared to mock transfection control by two folds (p<0.05) or more. Majority of the significant genes with altered expression clustered into immune-associated, signal transduction, and metabolic process categories. Differential gene expression of functionally important genes in these categories was also validated by real-time RT-PCR based relative gene-expression analysis. To our knowledge, this is the first report of in vitro replicon transfected RNA-Seq based transcriptome analysis to understand the host responses against HEV and HBV. PMID:24505321

  5. Encoded library technology screening of hepatitis C virus NS4B yields a small-molecule compound series with in vitro replicon activity.

    PubMed

    Arico-Muendel, Christopher; Zhu, Zhengrong; Dickson, Hamilton; Parks, Derek; Keicher, Jesse; Deng, Jianghe; Aquilani, Leah; Coppo, Frank; Graybill, Todd; Lind, Kenneth; Peat, Andrew; Thomson, Michael

    2015-01-01

    To identify novel antivirals to the hepatitis C virus (HCV) NS4B protein, we utilized encoded library technology (ELT), which enables purified proteins not amenable to standard biochemical screening methods to be tested against large combinatorial libraries in a short period of time. We tested NS4B against several DNA-encoded combinatorial libraries (DEL) and identified a single DEL feature that was subsequently progressed to off-DNA synthesis. The most active of the initial synthesized compounds had 50% inhibitory concentrations (IC50s) of 50 to 130 nM in a NS4B radioligand binding assay and 300 to 500 nM in an HCV replicon assay. Chemical optimization yielded compounds with potencies as low as 20 nM in an HCV genotype 1b replicon assay, 500 nM against genotype 1a, and 5 μM against genotype 2a. Through testing against other genotypes and genotype 2a-1b chimeric replicons and from resistance passage using the genotype 1b replicon, we confirmed that these compounds were acting on the proposed first transmembrane region of NS4B. A single sequence change (F98L) was identified as responsible for resistance, and it was thought to largely explain the relative lack of potency of this series against genotype 2a. Unlike other published series that appear to interact with this region, we did not observe sensitivity to amino acid substitutions at positions 94 and 105. The discovery of this novel compound series highlights ELT as a valuable approach for identifying direct-acting antivirals to nonenzymatic targets. PMID:25824229

  6. Combinations of various CpG motifs cloned into plasmid backbone modulate and enhance protective immunity of viral replicon DNA anthrax vaccines.

    PubMed

    Yu, Yun-Zhou; Ma, Yao; Xu, Wen-Hui; Wang, Shuang; Sun, Zhi-Wei

    2015-08-01

    DNA vaccines are generally weak stimulators of the immune system. Fortunately, their efficacy can be improved using a viral replicon vector or by the addition of immunostimulatory CpG motifs, although the design of these engineered DNA vectors requires optimization. Our results clearly suggest that multiple copies of three types of CpG motifs or combinations of various types of CpG motifs cloned into a viral replicon vector backbone with strong immunostimulatory activities on human PBMC are efficient adjuvants for these DNA vaccines to modulate and enhance protective immunity against anthrax, although modifications with these different CpG forms in vivo elicited inconsistent immune response profiles. Modification with more copies of CpG motifs elicited more potent adjuvant effects leading to the generation of enhanced immunity, which indicated a CpG motif dose-dependent enhancement of antigen-specific immune responses. Notably, the enhanced and/or synchronous adjuvant effects were observed in modification with combinations of two different types of CpG motifs, which provides not only a contribution to the knowledge base on the adjuvant activities of CpG motifs combinations but also implications for the rational design of optimal DNA vaccines with combinations of CpG motifs as "built-in" adjuvants. We describe an efficient strategy to design and optimize DNA vaccines by the addition of combined immunostimulatory CpG motifs in a viral replicon DNA plasmid to produce strong immune responses, which indicates that the CpG-modified viral replicon DNA plasmid may be desirable for use as vector of DNA vaccines. PMID:25265876

  7. Proteome Analysis of Liver Cells Expressing a Full- Length Hepatitis C Virus (HCV) Replicon and Biopsy Specimens of Posttransplantation Liver from HCV-Infected Patients

    SciTech Connect

    Jacobs, Jon M.; Diamond, Deborah L.; Chan, Eric Y.; Gritsenko, Marina A.; Qian, Weijun; Stastna, Miroslava; Baas, Tracey; Camp, David G.; Carithers, Jr., Robert L.; Smith, Richard D.; Katze, Michael G.

    2005-06-01

    The development of a reproducible model system for the study of Hepatitis C virus (HCV) infection has the potential to significantly enhance the study of virus-host interactions and provide future direction for modeling the pathogenesis of HCV. While there are studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been characterized. We report the first large scale proteome analysis of the highly permissive Huh-7.5 cell line containing a full length HCV replicon. We detected > 4,400 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled to mass spectrometry (MS). The set of Huh-7.5 proteins confidently identified is, to our knowledge, the most comprehensive yet reported for a human cell line. Consistent with the literature, a comparison of Huh-7.5 cells (+) and (-) the HCV replicon identified expression changes of proteins involved in lipid metabolism. We extended these analyses to liver biopsy material from HCV-infected patients where > 1,500 proteins were detected from 2 {micro}g protein lysate using the Huh-7.5 protein database and the accurate mass and time (AMT) tag strategy. These findings demonstrate the utility of multidimensional proteome analysis of the HCV replicon model system for assisting the determination of proteins/pathways affected by HCV infection. Our ability to extend these analyses to the highly complex proteome of small liver biopsies with limiting protein yields offers the unique opportunity to begin evaluating the clinical significance of protein expression changes associated with HCV infection.

  8. Use of fluorescence-activated vesicle sorting for isolation of Naked2-associated, basolaterally targeted exocytic vesicles for proteomics analysis.

    PubMed

    Cao, Zheng; Li, Cunxi; Higginbotham, James N; Franklin, Jeffrey L; Tabb, David L; Graves-Deal, Ramona; Hill, Salisha; Cheek, Kristin; Jerome, W Gray; Lapierre, Lynne A; Goldenring, James R; Ham, Amy-Joan L; Coffey, Robert J

    2008-09-01

    By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-alpha (TGFalpha), Naked2 coats TGFalpha-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGFalpha-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na(+)/K(+)-ATPase alpha1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis. PMID:18504258

  9. Molecular and Phenotypic Characterization of a Highly Evolved Type 2 Vaccine-Derived Poliovirus Isolated from Seawater in Brazil, 2014

    PubMed Central

    Cassemiro, Klécia Marília S. de Melo; Burlandy, Fernanda M.; Barbosa, Mikaela R. F.; Chen, Qi; Jorba, Jaume; Hachich, Elayse M.; Sato, Maria I. Z.; Burns, Cara C.; da Silva, Edson E.

    2016-01-01

    A type 2 vaccine-derived poliovirus (VDPV), differing from the Sabin 2 strain at 8.6% (78/903) of VP1 nucleotide positions, was isolated from seawater collected from a seaport in São Paulo State, Brazil. The P1/capsid region is related to the Sabin 2 strain, but sequences within the 5'-untranslated region and downstream of the P1 region were derived from recombination with other members of Human Enterovirus Species C (HEV-C). The two known attenuating mutations had reverted to wild-type (A481G in the 5'-UTR and Ile143Thr in VP1). The VDPV isolate had lost the temperature sensitive phenotype and had accumulated amino acid substitutions in neutralizing antigenic (NAg) sites 3a and 3b. The date of the initiating OPV dose, estimated from the number of synonymous substitutions in the capsid region, was approximately 8.5 years before seawater sampling, a finding consistent with a long time of virus replication and possible transmission among several individuals. Although no closely related type 2 VDPVs were detected in Brazil or elsewhere, this VDPV was found in an area with a mobile population, where conditions may favor both viral infection and spread. Environmental surveillance serves as an important tool for sensitive and early detection of circulating poliovirus in the final stages of global polio eradication. PMID:27019095

  10. Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: Inactivation of specific transcription factors

    SciTech Connect

    Fradkin, L.G.; Yoshinaga, S.K.; Berk, A.J.; Dasgupta, A.

    1987-11-01

    The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoted, however, was not altered by infection of cells with the virus. The authors conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirtus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.

  11. Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

    PubMed Central

    Flather, Dylan; Cathcart, Andrea L.; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D.; Semler, Bert L.

    2016-01-01

    Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

  12. Human Immunodeficiency Virus Tat-Activated Expression of Poliovirus Protein 2A Inhibits mRNA Translation

    NASA Astrophysics Data System (ADS)

    Sun, Xiao-Hong; Baltimore, David

    1989-04-01

    To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was contransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

  13. Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region.

    PubMed Central

    Bienz, K; Egger, D; Troxler, M; Pasamontes, L

    1990-01-01

    Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but did not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed. Images PMID:2154600

  14. Isolation of enteroviruses from water, suspended solids, and sediments from Galveston Bay: survival of poliovirus and rotavirus adsorbed to sediments.

    PubMed Central

    Rao, V C; Seidel, K M; Goyal, S M; Metcalf, T G; Melnick, J L

    1984-01-01

    The distribution and quantitation of enteroviruses among water, suspended solids, and compact sediments in a polluted estuary are described. Samples were collected sequentially from water, suspended solids, fluffy sediments (uppermost layer of bottom sediments), and compact sediment. A total of 103 samples were examined of which 27 (26%) were positive for virus. Polioviruses were recovered most often, followed by coxsackie B viruses and echoviruses 7 and 29. Virus was found most often attached to suspended solids: 72% of these samples were positive, whereas only 14% of water samples without solids yielded virus. Fluffy sediments yielded virus in 47% of the samples, whereas only 5% of compact bottom-sediment samples were positive. When associated with solids, poliovirus and rotavirus retained their infectious quality for 19 days. The same viruses remained infectious for only 9 days when freely suspended in seawater. Collection of suspended solids at ambient water pH appears to be very useful for the detection of virus; it has advantages over collecting and processing large volumes of water, with accompanying pH adjustment and salt addition for processing. PMID:6091548

  15. A Cysteine-Rich Motif in Poliovirus Protein 2CATPase Is Involved in RNA Replication and Binds Zinc In Vitro

    PubMed Central

    Pfister, Thomas; Jones, Keith W.; Wimmer, Eckard

    2000-01-01

    Protein 2CATPase of picornaviruses is involved in the rearrangement of host cell organelles, viral RNA replication, and encapsidation. However, the biochemical and molecular mechanisms by which 2CATPase engages in these processes are not known. To characterize functional domains of 2CATPase, we have focused on a cysteine-rich motif near the carboxy terminus of poliovirus 2CATPase. This region, which is well conserved among enteroviruses and rhinoviruses displaying an amino acid arrangement resembling zinc finger motifs, was studied by genetic and biochemical analyses. A mutation that replaced the first cysteine residue of the motif with a serine was lethal. A mutant virus which lacked the second of four potential coordination sites for zinc was temperature sensitive. At the restrictive temperature, RNA replication was inhibited whereas translation and polyprotein processing, assayed in vitro and in vivo, appeared to be normal. An intragenomic second-site revertant which reinserted the missing coordination site for zinc and recovered RNA replication at the restrictive temperature was isolated. The cysteine-rich motif was sufficient to bind zinc in vitro, as assessed in the presence of 4-(2-pyridylazo)resorcinol by a colorimetric assay. Zinc binding, however, was not required for hydrolysis of ATP. 2CATPase as well as its precursors 2BC and P2 were found to exist in a reduced form in poliovirus-infected cells. PMID:10590122

  16. Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

    DOE PAGESBeta

    Groom, Joseph; Chung, Daehwan; Olson, Daniel G.; Lynd, Lee R.; Guss, Adam M.; Westpheling, Janet

    2016-01-29

    Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hyperthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproduciblymore » transformed C. thermocellum at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. Lastly, these results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.« less

  17. Risk assessment, risk management and risk-based monitoring following a reported accidental release of poliovirus in Belgium, September to November 2014.

    PubMed

    Duizer, Erwin; Rutjes, Saskia; de Roda Husman, Ana Maria; Schijven, Jack

    2016-01-01

    On 6 September 2014, the accidental release of 10(13) infectious wild poliovirus type 3 (WPV3) particles by a vaccine production plant in Belgium was reported. WPV3 was released into the sewage system and discharged directly to a wastewater treatment plant (WWTP) and subsequently into rivers that flowed to the Western Scheldt and the North Sea. No poliovirus was detected in samples from the WWTP, surface waters, mussels or sewage from the Netherlands. Quantitative microbial risk assessment (QMRA) showed that the infection risks resulting from swimming in Belgium waters were above 50% for several days and that the infection risk by consuming shellfish harvested in the eastern part of the Western Scheldt warranted a shellfish cooking advice. We conclude that the reported release of WPV3 has neither resulted in detectable levels of poliovirus in any of the samples nor in poliovirus circulation in the Netherlands. This QMRA showed that relevant data on water flows were not readily available and that prior assumptions on dilution factors were overestimated. A QMRA should have been performed by all vaccine production facilities before starting up large-scale culture of WPV to be able to implement effective interventions when an accident happens. PMID:27020766

  18. Development of a multiplex RT-PCR assay for the identification of recombination types at different genomic regions of vaccine-derived polioviruses.

    PubMed

    Dimitriou, T G; Kyriakopoulou, Z; Tsakogiannis, D; Fikatas, A; Gartzonika, C; Levidiotou-Stefanou, S; Markoulatos, P

    2016-08-01

    Polioviruses (PVs) are the causal agents of acute paralytic poliomyelitis. Since the 1960s, poliomyelitis has been effectively controlled by the use of two vaccines containing all three serotypes of PVs, the inactivated poliovirus vaccine and the live attenuated oral poliovirus vaccine (OPV). Despite the success of OPV in polio eradication programme, a significant disadvantage was revealed: the emergence of vaccine-associated paralytic poliomyelitis (VAPP). VAPP is the result of accumulated mutations and putative recombination events located at the genome of attenuated vaccine Sabin strains. In the present study, ten Sabin isolates derived from OPV vaccinees and environmental samples were studied in order to identify recombination types located from VP1 to 3D genomic regions of virus genome. The experimental procedure that was followed was virus RNA extraction, reverse transcription to convert the virus genome into cDNA, PCR and multiplex-PCR using specific designed primers able to localize and identify each recombination following agarose gel electrophoresis. This multiplex RT-PCR assay allows for the immediate detection and identification of multiple recombination types located at the viral genome of OPV derivatives. After the eradication of wild PVs, the remaining sources of poliovirus infection worldwide would be the OPV derivatives. As a consequence, the immediate detection and molecular characterization of recombinant derivatives are important to avoid epidemics due to the circulation of neurovirulent viral strains. PMID:27098645

  19. An Alphavirus Replicon-Based Human Metapneumovirus Vaccine Is Immunogenic and Protective in Mice and Cotton Rats▿

    PubMed Central

    Mok, Hoyin; Tollefson, Sharon J.; Podsiad, Amy B.; Shepherd, Bryan E.; Polosukhin, Vasiliy V.; Johnston, Robert E.; Williams, John V.; Crowe, James E.

    2008-01-01

    Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that causes upper and lower respiratory tract infections in infants, the elderly, and immunocompromised individuals worldwide. Here, we developed Venezuelan equine encephalitis virus replicon particles (VRPs) encoding hMPV fusion (F) or attachment (G) glycoproteins and evaluated the immunogenicity and protective efficacy of these vaccine candidates in mice and cotton rats. VRPs encoding hMPV F protein, when administered intranasally, induced F-specific virus-neutralizing antibodies in serum and immunoglobulin A (IgA) antibodies in secretions at the respiratory mucosa. Challenge virus replication was reduced significantly in both the upper and lower respiratory tracts following intranasal hMPV challenge in these animals. However, vaccination with hMPV G protein VRPs did not induce neutralizing antibodies or protect animals from hMPV challenge. Close examination of the histopathology of the lungs of VRP-MPV F-vaccinated animals following hMPV challenge revealed no enhancement of inflammation or mucus production. Aberrant cytokine gene expression was not detected in these animals. Together, these results represent an important first step toward the use of VRPs encoding hMPV F proteins as a prophylactic vaccine for hMPV. PMID:18786987

  20. Profiling of antimicrobial resistance and plasmid replicon types in β-lactamase producing Escherichia coli isolated from Korean beef cattle

    PubMed Central

    Shin, Seung Won; Jung, Myunghwan; Shin, Min-Kyung

    2015-01-01

    In this study, 78 isolates of Escherichia coli isolated from Korean beef cattle farms were investigated for the production of extended-spectrum β-lactamase (ESBL) and/or AmpC β-lactamase. In the disc diffusion test with ampicillin, amoxicillin, cephalothin, ceftiofur, cefotaxime, ceftazidime, and cefoxitin, 38.5% of the isolates showed resistance to all of ampicillin, amoxicillin, and cephalothin. The double disc synergy method revealed that none of the isolates produced ESBL or AmpC β-lactamases. DNA sequencing showed that all isolates encoded genes for TEM-1-type β-lactamase. Moreover, 78.2% of the isolates transferred the TEM-1-type β-lactamase gene via conjugation. In plasmid replicon typing of all donors, IncFIB and IncFIA were identified in 71.4% and 41.0% of plasmids, respectively. In transconjugants, IncFIB and IncFIA were the most frequent types detected (61.5% and 41.0%, respectively). Overall, the present study indicates that selection pressures of antimicrobials on β-lactamases in beef cattle may be low relative to other livestock animals in Korea. Moreover, to reduce selection pressure and dissemination of β-lactamase, the long-term surveillance of antimicrobial use in domestic beef cattle should be established. PMID:26119172

  1. Development of a single-replicon miniBYV vector for co-expression of heterologous proteins.

    PubMed

    Prokhnevsky, Alex; Mamedov, Tarlan; Leffet, Brett; Rahimova, Rahila; Ghosh, Ananya; Mett, Vadim; Yusibov, Vidadi

    2015-02-01

    In planta production of recombinant proteins, including vaccine antigens and monoclonal antibodies, continues gaining acceptance. With the broadening range of target proteins, the need for vectors with higher performance is increasing. Here, we have developed a single-replicon vector based on beet yellows virus (BYV) that enables co-delivery of two target genes into the same host cell, resulting in transient expression of each target. This BYV vector maintained genetic stability during systemic spread throughout the host plant, Nicotiana benthamiana. Furthermore, we have engineered a miniBYV vector carrying the sequences encoding heavy and light chains of a monoclonal antibody (mAb) against protective antigen (PA) of Bacillius anthracis, and achieved the expression of the full-length functional anti-PA mAb at ~300 mg/kg of fresh leaf tissue. To demonstrate co-expression and functionality of two independent proteins, we cloned the sequences of the Pfs48/45 protein of Plasmodium falciparum and endoglycosidase F (PNGase F) from Flavobacterium meningosepticum into the miniBYV vector under the control of two subgenomic RNA promoters. Agroinfiltration of N. benthamiana with this miniBYV vector resulted in accumulation of biologically active Pfs48/45 that was devoid of N-linked glycosylation and had correct conformation and epitope display. Overall, our findings demonstrate that the new BYV-based vector is capable of co-expressing two functionally active recombinant proteins within the same host cell. PMID:25280556

  2. Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

    SciTech Connect

    Zheng Min; Jin Ningyi; Liu Qi; Huo Xiaowei; Li Yang; Hu Bo; Ma Haili; Zhu Zhanbo; Cong Yanzhao; Li Xiao; Jin Minglan; Zhu Guangze

    2009-08-15

    Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.

  3. Single-Cell Analysis Uncovers Extensive Biological Noise in Poliovirus Replication

    PubMed Central

    Schulte, Michael B.

    2014-01-01

    ABSTRACT Viral infections often begin with a very small number of initiating particles. Accordingly, the outcome of an infection is likely to be affected by variability in the initial molecular interactions between virus and host. In this study, we investigated the range of outcomes upon infection of single cells. We isolated individual cells infected with poliovirus at low or high multiplicities of infection (MOI) and measured viral genomic replication and infectious viral progeny in each cell. We first determined that at 7 h postinfection, the ratio of positive to negative strands in individual cells varies from 5:1 to more than 190:1, with and average of 20:1, suggesting a significant variability in RNA synthesis. We further found that while virus genome production is higher in cells infected at a high multiplicity, the production of infectious particles is largely independent of the number of viruses infecting each cell. Strikingly, by correlating RNA and particle production within individual infections, we uncovered a significant contribution of stochastic noise to the outcome of infection. At low MOI, stochastic influences appear as kinetic effects which are most critical at the initial steps in infection. At high MOI, stochastic influences appear to dictate the virus's ability to harness cellular resources. We conclude that biological noise is a critical determinant of the overall productivity of viral infections. The distinct nature of stochasticity in the outcome of infection by low and high numbers of viral particles may have important implications for our understanding of the determinants of successful viral infections. IMPORTANCE By correlating genome and particle production in single-cell infections, we elucidated sources of noise in viral infections. When a cell was infected by only a single infectious particle, variation in the kinetics of the initial steps of replication contributed significantly to the overall productivity of the infection

  4. Investigation et riposte à une épidémie de poliovirus sauvage à Kinshasa

    PubMed Central

    Nsambu, Muriel Nzazi; Bazira, Léodegal; Coulibaly, Tiekoura; Mbule, Albert; Wilmet, Michèle Dramaix; Likwela, Joris Losimba

    2013-01-01

    Introduction La République Démocratique du Congo a été considérée comme un pays à circulation rétablie de poliovirus sauvage (PVS). Cet article décrit l’épidémie de PVS qui a sévit dans la province de Kinshasa de 2010 à 2011. Méthodes Les analyses ont porté sur les cas de paralysie flasque aigüe (PFA) enregistrés de décembre 2010 à décembre 2011, les données de surveillance des PFA, les données de couverture vaccinale et celles du monitorage indépendant des activités de vaccination supplémentaires. Résultats Entre décembre 2010 à décembre 2011, 298 cas de PFA ont été enregistrés par les zones de santé parmi lesquels 34 cas de PVS confirmés. 58% des cas de PVS avaient plus de 15 ans avec plus d'hommes que de femmes. 10 passages d'activités de vaccination supplémentaires ont été mis en œuvre dont 4 avaient ciblé toute la population de Kinshasa. Il n'y a plus eu de cas de PVS après le 3e passage. Le monitorage des activités de vaccination a montré une proportion de sujets non vaccinés allant de 4 à 13%. La performance du système de surveillance était globalement bonne. Conclusion La prédominance des adultes parmi les cas notifiés traduit leur susceptibilité alors qu'ils ne sont généralement pas concernés lors des campagnes de vaccination supplémentaires. Ceci devrait engager les autorités sanitaires à envisager des activités vaccinales supplémentaires ciblant les adultes afin de casser plus rapidement la chaîne de transmission. Les faiblesses subsistant dans le système de surveillance pourraient être jugulées par le renforcement de la surveillance à base communautaire. PMID:24062866

  5. Combined immunization of infants with oral and inactivated poliovirus vaccines: results of a randomized trial in The Gambia, Oman, and Thailand. WHO Collaborative Study Group on Oral and Inactivated Poliovirus Vaccines.

    PubMed Central

    1996-01-01

    To assess an immunization schedule combining oral (OPV) and inactivated poliovirus vaccines (IPV), we conducted a clinical trial in the Gambia, Oman, and Thailand. Children were randomized to receive one of the following schedules: OPV at birth, 6, 10, and 14 weeks of age; OPV at birth followed by both OPV and IPV at 6, 10, and 14 weeks of age: or placebo at birth followed by IPV at 6, 10, and 14 weeks of age. A total of 1685 infants were enrolled; 24-week serum specimens were available for 1291 infants (77%). Across the study sites at 24 weeks of age, the proportion of seropositive children in the combined schedule group was 95-99% for type 1, 99-100% for type 2, and 97-100% for type 3. In the Gambia and Oman, the combined schedule performed significantly better than OPV for type 1 (95-97% versus 88-90%) and type 3 (97-99% versus 72-73%). In the Gambia and Oman, seroprevalences in the IPV group were lower for type 1 (significantly lower in the Gambia); significantly lower for type 2; and significantly higher for type 3, compared with the OPV group. In Thailand, the IPV group had significantly lower proportions of children who were seropositive for each of the three types, compared with the OPV group. The responses to OPV in the Gambia, Oman, and Thailand were consistent with previous studies from these countries. IPV given at 6, 10, and 14 weeks of age provided inadequate serological protection against poliovirus, especially type 1. The combined schedule provided the highest levels of serum antibody response, with mucosal immunity equivalent to that produced by OPV alone. PMID:8789924

  6. Growth performance of crossbred naked neck and normal feathered laying hens kept in tropical villages.

    PubMed

    Adomako, K; Olympio, O S; Hagan, J K; Hamidu, J A

    2014-01-01

    1. Two experiments were conducted to develop naked neck (Na/na) and normal feathered (na/na) crossbreds and compare their growth performance, linear body measurements and carcass characteristics in the first and second filial generations. 2. In the first experiment, 4 indigenous naked neck males (Na/na) were mated to 36 Lohmann commercial females (na/na) in a ratio of 1:9. The two genotypes (Na/na, na/na) were allocated randomly according to batches of hatch, sire lines and sex to three different villages. 3. In the second experiment, 10 males and 100 females of F1 Na/na birds were selected and mated inter se in a ratio of 1:10. The three genotypes (Na/Na, Na/na and na/na) were compared in a randomised complete block design experiment, with the three villages, hatch and sex as blocks and the three genotypes as treatments. F1 Na/na birds had significantly higher (P < 0.05) feed conversion ratio, body weight, body weight gain, linear body measurements, survivability and carcass yield than their na/na counterparts. 4. In the F2 generation, Na/Na and Na/na birds had significantly higher (P < 0.05) feed conversion ratio, body weight, body weight gain, linear body measurements, survivability and carcass yield compared to their na/na counterparts. 5. The birds showing the naked neck phenotype appeared to show superior performance compared to normal feathered birds and could be exploited for potential utilisation in local poultry production. PMID:25192492

  7. Naked plasmid DNA formulation: effect of different disaccharides on stability after lyophilisation.

    PubMed

    Quaak, Susanne G L; Haanen, John B A G; Beijnen, Jos H; Nuijen, Bastiaan

    2010-03-01

    Since plasmid DNA (pDNA) is unstable in solution, lyophilisation can be used to increase product shelf life. To prevent stress on pDNA molecules during lyophilisation, cryo- and lyoprotectants have to be added to the formulation. This study assessed the effect of disaccharides on naked pDNA stability after lyophilisation using accelerated stability studies. Naked pDNA was lyophilised with sucrose, trehalose, maltose or lactose in an excipient/DNA w/w ratio of 20. To one part of the vials extra residual moisture was introduced by placing the vials half opened in a 25 degrees C/60% RH climate chamber, before placing all vials in climate chambers (25 degrees C/60% RH and 40 degrees C/75% RH) for stability studies. An ex vivo human skin model was used to assess the effect of disaccharides on transfection efficiency. Lyophilisation resulted in amorphous cakes for all disaccharides with a residual water content of 0.8% w/w. Storage at 40 degrees C/75% RH resulted in decreasing supercoiled (SC) purity levels (sucrose and trehalose maintained approximately 80% SC purity), but not in physical collapse. The addition of residual moisture (values between 7.5% and 10% w/w) resulted in rapid collapse except for trehalose and decreasing SC purity for all formulations. In a separate experiment disaccharide formulation solutions show a slight but significant reduction (<3% with sucrose and maltose) in transfection efficiency when compared to pDNA dissolved in water. We demonstrate that disaccharides, like sucrose and trehalose, are effective lyoprotectants for naked pDNA. PMID:20204715

  8. Elimination of Specific miRNAs by Naked 14-nt sgRNAs

    PubMed Central

    Abe, Mayumi; Sato, Mari; Yoshida, Tetsuo; Yamada, Yoji; Tamura, Masato; Nashimoto, Masayuki

    2012-01-01

    tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is a newly developed technology to suppress mammalian gene expression. TRUE gene silencing works on the basis of a unique enzymatic property of mammalian tRNase ZL, which is that it can recognize a pre-tRNA-like or micro-pre-tRNA-like complex formed between target RNA and artificial small guide RNA (sgRNA) and can cleave any target RNA at any desired site. There are four types of sgRNA, 5′-half-tRNA, RNA heptamer, hook RNA, and ∼14-nt linear RNA. Here we show that a 14-nt linear-type sgRNA against human miR-16 can guide tRNase ZL cleavage of miR-16 in vitro and can downregulate the miR-16 level in HEK293 cells. We also demonstrate that the 14-nt sgRNA can be efficiently taken up without any transfection reagents by living cells and can exist stably in there for at least 24 hours. The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells. Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines. Our observations suggest that in general we can eliminate a specific cellular miRNA at least by ∼50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing. PMID:22675569

  9. Multidimensional MRI-CT atlas of the naked mole-rat brain (Heterocephalus glaber).

    PubMed

    Seki, Fumiko; Hikishima, Keigo; Nambu, Sanae; Okanoya, Kazuo; Okano, Hirotaka J; Sasaki, Erika; Miura, Kyoko; Okano, Hideyuki

    2013-01-01

    Naked mole-rats have a variety of distinctive features such as the organization of a hierarchical society (known as eusociality), extraordinary longevity, and cancer resistance; thus, it would be worthwhile investigating these animals in detail. One important task is the preparation of a brain atlas database that provide comprehensive information containing multidimensional data with various image contrasts, which can be achievable using a magnetic resonance imaging (MRI). Advanced MRI techniques such as diffusion tensor imaging (DTI), which generates high contrast images of fiber structures, can characterize unique morphological properties in addition to conventional MRI. To obtain high spatial resolution images, MR histology, DTI, and X-ray computed tomography were performed on the fixed adult brain. Skull and brain structures were segmented as well as reconstructed in stereotaxic coordinates. Data were also acquired for the neonatal brain to allow developmental changes to be observed. Moreover, in vivo imaging of naked mole-rats was established as an evaluation tool of live animals. The data obtained comprised three-dimensional (3D) images with high tissue contrast as well as stereotaxic coordinates. Developmental differences in the visual system were highlighted in particular by DTI. Although it was difficult to delineate optic nerves in the mature adult brain, parts of them could be distinguished in the immature neonatal brain. From observation of cortical thickness, possibility of high somatosensory system development replaced to the visual system was indicated. 3D visualization of brain structures in the atlas as well as the establishment of in vivo imaging would promote neuroimaging researches towards detection of novel characteristics of eusocial naked mole-rats. PMID:24391551

  10. Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors

    SciTech Connect

    Filone, Claire Marie; Heise, Mark; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Bertolotti-Ciarlet, Andrea . E-mail: aciarlet@mail.med.upenn.edu

    2006-12-20

    Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by {beta}-galactosidase {alpha}-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.

  11. Incompatibility of Lactobacillus Vectors with Replicons Derived from Small Cryptic Lactobacillus Plasmids and Segregational Instability of the Introduced Vectors

    PubMed Central

    Posno, M.; Leer, R. J.; van Luijk, N.; van Giezen, M. J. F.; Heuvelmans, P. T. H. M.; Lokman, B. C.; Pouwels, P. H.

    1991-01-01

    Three new Lactobacillus vectors based on cryptic Lactobacillus plasmids were constructed. The shuttle vector pLP3537 consists of a 2.3-kb plasmid from Lactobacillus pentosus MD353, an erythromycin resistance gene from Staphylococcus aureus plasmid pE194, and pUC19 as a replicon for Escherichia coli. The vectors pLPE317 and pLPE323, which do not contain E. coli sequences, were generated by introducing the erythromycin resistance gene of pE194 into a 1.7- and a 2.3-kb plasmid from L. pentosus MD353, respectively. These vectors and the shuttle vector pLP825 (M. Posno, R. J. Leer, J. M. M. van Rijn, B. C. Lokman, and P. H. Pouwels, p. 397-401, in A. T. Ganesan and J. A. Hoch, ed., Genetics and biotechnology of bacilli, vol. 2, 1988) could be introduced by electroporation into Lactobacillus casei, L. pentosus, L. plantarum, L. acidophilus, L. fermentum, and L. brevis strains with similar efficiencies. Transformation efficiencies were strain dependent and varied from 102 to 107 transformants per μg of DNA. Plasmid DNA analysis of L. pentosus MD353 transformants revealed that the introduction of pLP3537 or pLPE323 was invariably accompanied by loss of the endogenous 2.3-kb plasmid. Remarkably, pLPE317 could only be introduced into an L. pentosus MD353 strain that had been previously cured of its endogenous 1.7-kb plasmid. The curing phenomena are most likely to be explained by the incompatibility of the vectors and resident plasmids. Lactobacillus vectors are generally rapidly lost when cells are cultivated in the absence of selective pressure. However, pLPE323 is stable in three of four Lactobacillus strains tested so far. Images PMID:16348515

  12. Dew inspired breathing-based detection of genetic point mutation visualized by naked eye

    NASA Astrophysics Data System (ADS)

    Xie, Liping; Wang, Tongzhou; Huang, Tianqi; Hou, Wei; Huang, Guoliang; Du, Yanan

    2014-09-01

    A novel label-free method based on breathing-induced vapor condensation was developed for detection of genetic point mutation. The dew-inspired detection was realized by integration of target-induced DNA ligation with rolling circle amplification (RCA). The vapor condensation induced by breathing transduced the RCA-amplified variances in DNA contents into visible contrast. The image could be recorded by a cell phone for further or even remote analysis. This green assay offers a naked-eye-reading method potentially applied for point-of-care liver cancer diagnosis in resource-limited regions.

  13. Sequence selective naked-eye detection of DNA harnessing extension of oligonucleotide-modified nucleotides.

    PubMed

    Verga, Daniela; Welter, Moritz; Marx, Andreas

    2016-02-01

    DNA polymerases can efficiently and sequence selectively incorporate oligonucleotide (ODN)-modified nucleotides and the incorporated oligonucleotide strand can be employed as primer in rolling circle amplification (RCA). The effective amplification of the DNA primer by Φ29 DNA polymerase allows the sequence-selective hybridisation of the amplified strand with a G-quadruplex DNA sequence that has horse radish peroxidase-like activity. Based on these findings we develop a system that allows DNA detection with single-base resolution by naked eye. PMID:26774580

  14. Origin of the bright prompt optical emission in the naked eye burst

    SciTech Connect

    Hascoeet, R.; Daigne, F.; Mochkovitch, R.

    2010-10-15

    The huge optical brightness of GRB 080319B (the 'Naked Eye Burst') makes this event really challenging for models of the prompt GRB emission. In the framework of the internal shock model, we investigate a scenario where the dominant radiative process is synchrotron emission and the high optical flux is due to the dynamical properties of the relativistic outflow : if the initial Lorentz factor distribution in the jet is highly variable, many internal shocks will form within the outflow at various radii. The most violent shocks will produce the main gamma-ray component while the less violent ones will contribute at lower energy, including the optical range.

  15. Detection of coronavirus genomes in Moluccan naked-backed fruit bats in Indonesia.

    PubMed

    Anindita, Paulina Duhita; Sasaki, Michihito; Setiyono, Agus; Handharyani, Ekowati; Orba, Yasuko; Kobayashi, Shintaro; Rahmadani, Ibnu; Taha, Siswatiana; Adiani, Sri; Subangkit, Mawar; Nakamura, Ichiro; Sawa, Hirofumi; Kimura, Takashi

    2015-04-01

    Bats have been shown to serve as natural reservoirs for numerous emerging viruses including severe acute respiratory syndrome coronavirus (SARS-CoV). In the present study, we report the discovery of bat CoV genes in Indonesian Moluccan naked-backed fruit bats (Dobsonia moluccensis). A partial RNA-dependent RNA polymerase gene sequence was detected in feces and tissues samples from the fruit bats, and the region between the RdRp and helicase genes could also be amplified from fecal samples. Phylogenetic analysis suggested that these bat CoVs are related to members of the genus Betacoronavirus. PMID:25643817

  16. Eliciting antigen-specific egg-yolk IgY with naked DNA.

    PubMed

    Romito, M; Viljoen, G J; Du Plessis, D H

    2001-09-01

    Immunization with naked DNA was used to elicit chicken egg yolk antibodies (IgY). Layer hens were inoculated with plasmid DNA encoding the enhanced green fluorescent protein, the fusion protein of Newcastle disease virus, and VP2 of African horse sickness virus. IgY was extracted from egg yolks by polyethylene glycol precipitation. Specific antibodies were present in the yolks of eggs from hens immunized with each of the three different plasmids. This approach to raising polyclonal antibodies obviates the need to produce and purify large quantities of proteins for immunization and can potentially yield large amounts of diagnostically or therapeutically useful reagents. PMID:11570510

  17. Dew inspired breathing-based detection of genetic point mutation visualized by naked eye

    PubMed Central

    Xie, Liping; Wang, Tongzhou; Huang, Tianqi; Hou, Wei; Huang, Guoliang; Du, Yanan

    2014-01-01

    A novel label-free method based on breathing-induced vapor condensation was developed for detection of genetic point mutation. The dew-inspired detection was realized by integration of target-induced DNA ligation with rolling circle amplification (RCA). The vapor condensation induced by breathing transduced the RCA-amplified variances in DNA contents into visible contrast. The image could be recorded by a cell phone for further or even remote analysis. This green assay offers a naked-eye-reading method potentially applied for point-of-care liver cancer diagnosis in resource-limited regions. PMID:25199907

  18. Data on macrophage mediated muscle transfection upon delivery of naked plasmid DNA with block copolymers.

    PubMed

    Mahajan, Vivek; Gaymalov, Zagit; Alakhova, Daria; Gupta, Richa; Zucker, Irving H; Kabanov, Alexander V

    2016-06-01

    The data contains 14 figures supporting the research article "Horizontal gene transfer from macrophages to ischemic muscles upon delivery of naked DNA with Pluronic block copolymers" [1]. The data explains the surgical procedure and histological characterization of Murine Hind Limb Ischemia. The data also shows the kinetics of luciferase gene expression, spread of GFP expression through muscle and the colocalization of GFP with cellular markers in ischemic muscles injected with pDNA alone or pDNA/Pluronic. Finally the data shows the effect of Pluronic Block Copolymer to enhance total gene expression (cmv-promoter driven luciferase gene) in coculture of DNA transfected MØs with muscle cells. PMID:27222845

  19. Spatiotemporal multiplexing method for visual field of view extension in holographic displays with naked eye observation

    NASA Astrophysics Data System (ADS)

    Finke, G.; Kujawińska, M.; Kozacki, T.; Zaperty, W.

    2016-09-01

    In this paper we propose a method which allows to overcome the basic functional problems in holographic displays with naked eye observation caused by delivering too small images visible in narrow viewing angles. The solution is based on combining the spatiotemporal multiplexing method with a 4f optical system. It enables to increase an aperture of a holographic display and extend the angular visual field of view. The applicability of the modified display is evidenced by Wigner distribution analysis of holographic imaging with spatiotemporal multiplexing method and by the experiments performed at the display demonstrator.

  20. Poliovirus infection of cyclophosphamide-treated mice results in persistence and late paralysis: I. Clinical, pathologic, and immunologic studies.

    PubMed

    Jubelt, B; Meagher, J B

    1984-04-01

    An attenuated human poliovirus infection of cyclophosphamide (CY)-treated mice was developed as a model of persistent CNS enterovirus infections and as an investigation of the interaction of virus with motor neurons during persistence. Ten percent of mice inoculated intracerebrally with undiluted virus developed clinical disease by day 90, but of those treated with CY, 80% developed disease. At higher virus dilutions plus CY there was a marked increase in the incubation period. The latest onset of clinical disease occurred on day 146. Only paralyzed animals had pathologic changes in the spinal cord and virus antigen in anterior horn cells. Neutralizing antibodies were suppressed by CY, as were humoral and cellular immune responses to other antigens. PMID:6322051

  1. A Single Amino Acid Substitution in Poliovirus Nonstructural Protein 2CATPase Causes Conditional Defects in Encapsidation and Uncoating

    PubMed Central

    Asare, Emmanuel; Mugavero, JoAnn; Jiang, Ping; Paul, Aniko V.

    2016-01-01

    ABSTRACT The specificity of encapsidation of C-cluster enteroviruses depends on an interaction between capsid proteins and nonstructural protein 2CATPase. In particular, residue N252 of poliovirus 2CATPase interacts with VP3 of coxsackievirus A20, in the context of a chimeric virus. Poliovirus 2CATPase has important roles both in RNA replication and encapsidation. In this study, we searched for additional sites in 2CATPase, near N252, that are required for encapsidation. Accordingly, segments adjacent to N252 were analyzed by combining triple and single alanine mutations to identify residues required for function. Two triple alanine mutants exhibited defects in RNA replication. The remaining two mutations, located in secondary structures in a predicted three-dimensional model of 2CATPase, caused lethal growth phenotypes. Most single alanine mutants, derived from the lethal variants, were either quasi-infectious and yielded variants with wild-type (wt) or temperature-sensitive (ts) growth phenotypes or had a lethal growth phenotype due to defective RNA replication. The K259A mutation, mapping to an α helix in the predicted structure of 2CATPase, resulted in a cold-sensitive virus. In vivo protein synthesis and virus production were strikingly delayed at 33°C relative to the wt, suggesting a defect in uncoating. Studies with a reporter virus indicated that this mutant is also defective in encapsidation at 33°C. Cell imaging confirmed a much-reduced production of K259A mature virus at 33°C relative to the wt. In conclusion, we have for the first time linked a cold-sensitive encapsidation defect in 2CATPase (K259A) to a subsequent delay in uncoating of the virus particle at 33°C during the next cycle of infection. IMPORTANCE Enterovirus morphogenesis, which involves the encapsidation of newly made virion RNA, is a process still poorly understood. Elucidation of this process is important for future drug development for a large variety of diseases caused by these

  2. A severe case of co-infection with Enterovirus 71 and vaccine-derived Poliovirus type II.

    PubMed

    Ma, Shaohui; Du, Zengqing; Feng, Min; Che, Yanchun; Li, Qihan

    2015-11-01

    Enterovirus 71 (EV71) is often identified as the primary pathogen that directly leads to severe cases of HFMD, whereas the association between other enteroviruses and EV71 infection remains largely unclear. Here we report a rare case of a 5-year-old boy co-infected with EV71 and vaccine-derived Poliovirus (VDPV) type II, which were identified based on PCR and sequence analysis results and clinical symptoms and were characterized on CT. We determined that the EV71 strain belongs to the C4 subtype, and the VDPV II strain was closely genetically related to the reference Sabin type II strain. This report may improved our understanding of the clinical significance of the associations between clinical signs and the infectious properties of the involved pathogens. PMID:26361010

  3. Progress toward Global Interruption of Wild Poliovirus Transmission, 2010–2013 and Tackling the Challenges to Complete Eradication

    PubMed Central

    Wassilak, Steven G.F.; Oberste, M. Steven; Tangermann, Rudolph H.; Diop, Ousmane M.; Jafari, Hamid S.; Armstrong, Gregory L.

    2015-01-01

    Despite substantial progress, global polio eradication has remained elusive. Indigenous wild poliovirus (WPV) transmission in four endemic countries (Afghanistan, India, Nigeria, and Pakistan) persisted into 2010 and outbreaks from imported WPV continued. By 2013, most outbreaks in the interim were promptly controlled. The number of polio-affected districts globally has declined by74% (from 481 in 2009 to 126 in 2013), including a 79% decrease in the number of affected districts in endemic countries (from 304 to 63). India is now polio-free. The challenges to success in the remaining polio-endemic countries include 1) threats to the security of vaccinators in each country and a ban on polio vaccination in areas of Afghanistan and Pakistan; 2) a risk of decreased government commitment; and 3) remaining surveillance gaps. Coordinated efforts under the International Health Regulations and efforts to mitigate the challenges provide a clear opportunity to soon secure global eradication. PMID:25316873

  4. Construction of a mutagenesis cartridge for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants

    SciTech Connect

    Kuhn, R.J.; Tada, H.; Ypma-Wong, M.F.; Dunn, J.J.; Semler, B.L.; Wimmer, E.

    1988-01-01

    By following a strategy of genetic analysis of poliovirus, the authors have constructed a synthetic mutagenesis cartridge spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type I (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed them to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alteration of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. They suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).

  5. Progress toward global interruption of wild poliovirus transmission, 2010-2013, and tackling the challenges to complete eradication.

    PubMed

    Wassilak, Steven G F; Oberste, M Steven; Tangermann, Rudolph H; Diop, Ousmane M; Jafari, Hamid S; Armstrong, Gregory L

    2014-11-01

    Despite substantial progress, global polio eradication has remained elusive. Indigenous wild poliovirus (WPV) transmission in 4 endemic countries (Afghanistan, India, Nigeria, and Pakistan) persisted into 2010 and outbreaks from imported WPV continued. By 2013, most outbreaks in the interim were promptly controlled. The number of polio-affected districts globally has declined by 74% (from 481 in 2009 to 126 in 2013), including a 79% decrease in the number of affected districts in endemic countries (from 304 to 63). India is now polio-free. The challenges to success in the remaining polio-endemic countries include (1) threats to the security of vaccinators in each country and a ban on polio vaccination in areas of Afghanistan and Pakistan; (2) a risk of decreased government commitment; and (3) remaining surveillance gaps. Coordinated efforts under the International Health Regulations and efforts to mitigate the challenges provide a clear opportunity to soon secure global eradication. PMID:25316873

  6. Modified procedure for extraction of poliovirus from naturally-infected oysters using Cat-Floc and beef extract

    SciTech Connect

    Landry, E.F.; Vaughn, J.M.; Vicale, T.J.

    1980-02-01

    Methods for recovery of poliovirus type 1 from naturally-infected oysters were examined. Extraction procedures analyzed included glycine-saline and polyelectrolyte (Cat-Floc) methods followed by concentration using modifications of an acid precipitation technique. Direct viral assay of shellfish homogenates, when compared to virus recovery following extraction, indicated that substantially fewer viruses were detected in initial homogenates. These data appeared to support the contention that input values based on homogenate assay were inappropriate in determining recovery efficiencies with naturally-infected shellfish. Since absolute efficiencies could not be determined, relative efficiencies using samples from pooled homogenates were used to determine the recovery efficiencies of various extraction procedures. Cat-Floc extraction followed by a beef extract-modified acid precipitation technique resulted in higher virus recoveries than a glycine-saline extraction procedure.

  7. Construction of recombinant DNA molecules by the use of a single stranded DNA generated by the polymerase chain reaction: its application to chimeric hepatitis A virus/poliovirus subgenomic cDNA.

    PubMed Central

    Wychowski, C; Emerson, S U; Silver, J; Feinstone, S M

    1990-01-01

    In order to study the importance of VP4 in picornavirus replication and translation, we replaced the hepatitis A virus (HAV) VP4 with the poliovirus (PV1) VP4. Using a modification of oligonucleotide site directed mutagenesis and the polymerase chain reaction (PCR), we created a subgenomic cDNA chimera of hepatitis A virus in which the precise sequences coding for HAV VP4 capsid protein were replaced by the sequences coding for the poliovirus VP4 capsid protein. The method involved the use of PCR primers corresponding to the 3' and 5' ends of the poliovirus VP4 sequence and that had HAV VP4 3' and 5' flanking sequences on their 5'ends. Single stranded DNA of 240 and 242 nt containing the 204 nt coding for the complete poliovirus VP4 were produced by using a limiting amount of one of the primers in a PCR reaction. These single stranded PCR products were used like mutagenic oligonucleotides on a single stranded phagemid containing the first 2070 bases of the HAV genome. Using this technique, we precisely replaced the HAV VP4 gene by the poliovirus VP4 gene as determined by DNA sequencing. The cDNA was transcribed into RNA and translated in vitro. The resulting protein could be precipitated by antibody to poliovirus VP4 but not to HAV VP4. Images PMID:2156236

  8. Replicon-free and markerless methods for genomic insertion of DNAs in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli.

    PubMed

    Chiang, Chung-Jen; Chen, Po Ting; Chao, Yun-Peng

    2008-12-01

    Genetic manipulation of cells for desired traits is the most appreciable strategy implemented in the field of bioengineering. However, this approach closely relies on the use of plasmids and is commonly afflicted by the potential problem of plasmid instability and safety caution. Meanwhile, it may also lead to the spread of antibiotic-resistant markers with replicons of plasmids to the environment. However, this issue has long been neglected. In this study, we have addressed these subjects by developing replicon-free and markerless methods for chromosomal insertion of genes and controlled expression of genomic genes in Escherichia coli. For the former application, the integration vectors of conditional replication were incorporated with the prophage attachment site and duplicated FRT sites. Their utility was illustrated by site-specific insertion of target genes, the endogenous dxs gene and three heterologous genes consisting of gps, crtI, and crtB, fused to T7 promoter into E. coli genome. For the latter application, the template vectors for promoter replacement were constructed to carry a DNA cassette containing the T7 promoter linked to a selective marker flanked with the FRT site. Subsequently, it was illustrated by replacement of the native promoter of chromosomal pckA by the T7 promoter. Finally, with the aid of FLP recombinase supplied from a helper plasmid, the regions containing replicon and/or selective markers in inserted DNAs were eliminated from integrants for both approaches. As a consequence, the expression of these five genes was subject to control by one response regulator, T7 RNA polymerase, in a regulon way, resulting in a high and stable production of lycopene in the cell. This result indicates the promise of developed methods for genome engineering in E. coli. PMID:18553504

  9. Natural selection of adaptive mutations in non-structural genes increases trans-encapsidation of hepatitis C virus replicons lacking envelope protein genes.

    PubMed

    Fournier, Carole; Helle, François; Descamps, Véronique; Morel, Virginie; François, Catherine; Dedeurwaerder, Sarah; Wychowski, Czeslaw; Duverlie, Gilles; Castelain, Sandrine

    2013-05-01

    A trans-packaging system for hepatitis C virus (HCV) replicons lacking envelope glycoproteins was developed. The replicons were efficiently encapsidated into infectious particles after expression in trans of homologous HCV envelope proteins under the control of an adenoviral vector. Interestingly, expression in trans of core or core, p7 and NS2 with envelope proteins did not enhance trans-encapsidation. Expression of heterologous envelope proteins, in the presence or absence of heterologous core, p7 and NS2, did not rescue single-round infectious particle production. To increase the titre of homologous, single-round infectious particles in our system, successive cycles of trans-encapsidation and infection were performed. Four cycles resulted in a 100-fold increase in the yield of particles. Sequence analysis revealed a total of 16 potential adaptive mutations in two independent experiments. Except for a core mutation in one experiment, all the mutations were located in non-structural regions mainly in NS5A (four in domain III and two near the junction with the NS5B gene). Reverse genetics studies suggested that D2437A and S2443T adaptive mutations, which are located at the NS5A-B cleavage site did not affect viral replication, but enhanced the single-round infectious particles assembly only in trans-encapsidation model. In conclusion, our trans-encapsidation system enables the production of HCV single-round infectious particles. This system is adaptable and can positively select variants. The adapted variants promote trans-encapsidation and should constitute a valuable tool in the development of replicon-based HCV vaccines. PMID:23288424

  10. Unique plasmids generated via pUC replicon mutagenesis in an error-prone thermophile derived from Geobacillus kaustophilus HTA426.

    PubMed

    Kobayashi, Jyumpei; Tanabiki, Misaki; Doi, Shohei; Kondo, Akihiko; Ohshiro, Takashi; Suzuki, Hirokazu

    2015-11-01

    The plasmid pGKE75-catA138T, which comprises pUC18 and the catA138T gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CATA138T), serves as an Escherichia coli-Geobacillus kaustophilus shuttle plasmid that confers moderate chloramphenicol resistance on G. kaustophilus HTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75-catA138T in an error-prone thermophile, generating the mutant plasmid pGKE75(αβ)-catA138T responsible for substantial chloramphenicol resistance at 65°C. pGKE75(αβ)-catA138T contained no mutation in the catA138T gene but had two mutations in the pUC replicon, even though the replicon has no apparent role in G. kaustophilus. Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75(αβ)-catA138T is attributable to increases in intracellular CATA138T and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75(αβ)-catA138T. The decrease in incomplete plasmids may be due to optimization of plasmid replication by RNA species transcribed from the mutant pUC replicon, which were actually produced in G. kaustophilus. It is noteworthy that G. kaustophilus was transformed with pGKE75(αβ)-catA138T using chloramphenicol selection at 60°C. In addition, a pUC18 derivative with the two mutations propagated in E. coli at a high copy number independently of the culture temperature and high plasmid stability. Since these properties have not been observed in known plasmids, the outcomes extend the genetic toolboxes for G. kaustophilus and E. coli. PMID:26319877

  11. Unique Plasmids Generated via pUC Replicon Mutagenesis in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

    PubMed Central

    Kobayashi, Jyumpei; Tanabiki, Misaki; Doi, Shohei; Kondo, Akihiko; Ohshiro, Takashi

    2015-01-01

    The plasmid pGKE75-catA138T, which comprises pUC18 and the catA138T gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CATA138T), serves as an Escherichia coli-Geobacillus kaustophilus shuttle plasmid that confers moderate chloramphenicol resistance on G. kaustophilus HTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75-catA138T in an error-prone thermophile, generating the mutant plasmid pGKE75αβ-catA138T responsible for substantial chloramphenicol resistance at 65°C. pGKE75αβ-catA138T contained no mutation in the catA138T gene but had two mutations in the pUC replicon, even though the replicon has no apparent role in G. kaustophilus. Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75αβ-catA138T is attributable to increases in intracellular CATA138T and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75αβ-catA138T. The decrease in incomplete plasmids may be due to optimization of plasmid replication by RNA species transcribed from the mutant pUC replicon, which were actually produced in G. kaustophilus. It is noteworthy that G. kaustophilus was transformed with pGKE75αβ-catA138T using chloramphenicol selection at 60°C. In addition, a pUC18 derivative with the two mutations propagated in E. coli at a high copy number independently of the culture temperature and high plasmid stability. Since these properties have not been observed in known plasmids, the outcomes extend the genetic toolboxes for G. kaustophilus and E. coli. PMID:26319877

  12. Motion of test particles in the field of a naked singularity

    NASA Astrophysics Data System (ADS)

    Boshkayev, K.; Gasperín, E.; Gutiérrez-Piñeres, A. C.; Quevedo, H.; Toktarbay, S.

    2016-01-01

    We investigate the motion of test particles in the gravitational field of a static naked singularity generated by a mass distribution with a quadrupole moment. We use the quadrupole metric (q metric) which is the simplest generalization of the Schwarzschild metric with a quadrupole parameter. We study the influence of the quadrupole on the motion of massive test particles and photons and show that the behavior of the geodesics can drastically depend on the values of the quadrupole parameter. In particular, we prove explicitly that the perihelion distance depends on the value of the quadrupole. Moreover, we show that an accretion disk on the equatorial plane of the quadrupole source can be either continuous or discrete, depending on the value of the quadrupole. The inner radius of the disk can be used in certain cases to determine the value of the quadrupole parameter. The case of a discrete accretion is interpreted as due to the presence of repulsive gravity generated by the naked singularity. Radial geodesics are also investigated and compared with the Schwarzschild counterparts.

  13. FLT3 Ligand as a Molecular Adjuvant for Naked RNA Vaccines.

    PubMed

    Kreiter, Sebastian; Diken, Mustafa; Selmi, Abderraouf; Petschenka, Jutta; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Intranodal immunization with antigen-encoding naked mRNA has proven to be an efficacious and safe approach to induce antitumor immunity. Thanks to its unique characteristics, mRNA can act not only as a source for antigen but also as an adjuvant for activation of the immune system. The search for additional adjuvants that can be combined with mRNA to further improve the potency of the immunization revealed Fms-like tyrosine kinase 3 (FLT3) ligand as a potent candidate. Systemic administration of the dendritic cell-activating FLT3 ligand prior to or along with mRNA immunization-enhanced priming and expansion of antigen-specific CD8(+) T cells in lymphoid organs, T-cell homing into melanoma tumors, and therapeutic activity of the intranodally administered mRNA. Both compounds demonstrate a successful combination in terms of boosting the immune response. This chapter describes methods for intranodal immunization with naked mRNA by co-administration of FLT3 ligand, which leads to strong synergistic effects. PMID:27236799

  14. Naked gene therapy of hepatocyte growth factor for dextran sulfate sodium-induced colitis in mice

    SciTech Connect

    Kanbe, Takamasa |; Murai, Rie; Mukoyama, Tomoyuki; Murawaki, Yoshiyuki |; Hashiguchi, Ko-ichi; Yoshida, Yoko; Tsuchiya, Hiroyuki; Kurimasa, Akihiro; Harada, Ken-ichi; Yashima, Kazuo; Nishimuki, Eiji; Shabana, Noriko; Kishimoto, Yukihiro; Kojyo, Haruhiko; Miura, Kunihiko; Kawasaki, Hironaka; Murawaki, Yoshikazu; Shiota, Goshi . E-mail: gshiota@grape.med.tottori-u.ac.jp

    2006-07-14

    Ulcerative colitis (UC) is progressive and relapsing disease. To explore the therapeutic effects of naked gene therapy of hepatocyte growth factor (HGF) on UC, the SR{alpha} promoter driving HGF gene was intrarectally administered to the mice in which colitis was induced by dextran sulfate sodium (DSS). Expression of the transgene was seen in surface epithelium, lamina propria, and muscularis mucosae. The HGF-treated mice showed reduced colonic mucosal damage and increased body weights, compared with control mice (P < 0.01 and P < 0.05, respectively). The HGF-treated mice displayed increased number of PCNA-positive cells and decreased number of apoptotic cells than in control mice (P < 0.01, each). Phosphorylated AKT was dramatically increased after HGF gene administration, however, phosphorylated ERK1/2 was not altered. Microarray analysis revealed that HGF induced expression of proliferation- and apoptosis-associated genes. These data suggest that naked HGF gene delivery causes therapeutic effects through regulation of many downstream genes.

  15. Naked singularity formation in the collapse of a spherical cloud of counterrotating particles

    NASA Astrophysics Data System (ADS)

    Harada, Tomohiro; Iguchi, Hideo; Nakao, Ken-Ichi

    1998-08-01

    We investigate the collapse of a spherical cloud of counterrotating particles. An explicit solution for metric functions is given using an elliptic integral. If the specific angular momentum L(r)=O(r2) at r-->0, no central singularity occurs. With L(r) like that, there is a finite region around the center that bounces. On the other hand, if the order of L(r) is higher than that, a central singularity occurs. In a marginally bound collapse with L(r)=4F(r), a naked singularity occurs, where F(r) is the Misner-Sharp mass. The solution for this case is expressed by elementary functions. For 40, there is a finite region around the center that bounces and a naked singularity occurs. For 0<=L/F<4 at r-->0, there is no such region. The results suggest that rotation may play a crucial role on the final fate of collapse.

  16. In vivo silencing of alpha-synuclein using naked siRNA

    PubMed Central

    Lewis, Jada; Melrose, Heather; Bumcrot, David; Hope, Andrew; Zehr, Cynthia; Lincoln, Sarah; Braithwaite, Adam; He, Zhen; Ogholikhan, Sina; Hinkle, Kelly; Kent, Caroline; Toudjarska, Ivanka; Charisse, Klaus; Braich, Ravi; Pandey, Rajendra K; Heckman, Michael; Maraganore, Demetrius M; Crook, Julia; Farrer, Matthew J

    2008-01-01

    Background Overexpression of α-synuclein (SNCA) in families with multiplication mutations causes parkinsonism and subsequent dementia, characterized by diffuse Lewy Body disease post-mortem. Genetic variability in SNCA contributes to risk of idiopathic Parkinson's disease (PD), possibly as a result of overexpression. SNCA downregulation is therefore a valid therapeutic target for PD. Results We have identified human and murine-specific siRNA molecules which reduce SNCA in vitro. As a proof of concept, we demonstrate that direct infusion of chemically modified (naked), murine-specific siRNA into the hippocampus significantly reduces SNCA levels. Reduction of SNCA in the hippocampus and cortex persists for a minimum of 1 week post-infusion with recovery nearing control levels by 3 weeks post-infusion. Conclusion We have developed naked gene-specific siRNAs that silence expression of SNCA in vivo. This approach may prove beneficial toward our understanding of the endogenous functional equilibrium of SNCA, its role in disease, and eventually as a therapeutic strategy for α-synucleinopathies resulting from SNCA overexpression. PMID:18976489

  17. A novel opal closest-packing photonic crystal for naked-eye glucose detection.

    PubMed

    Hong, Xiaodi; Peng, Yuan; Bai, Jialei; Ning, Baoan; Liu, Yuanyuan; Zhou, Zhijiang; Gao, Zhixian

    2014-04-01

    A novel opal closest-packing (OCP) photonic crystal (PC) is successfully prepared for naked-eye glucose detection. This PC is fabricated via a vertical convective self-assembly method with a new type of monodisperse microsphere polymerized by co-monomers, namely, methyl methacrylate (MMA), N-isopropylacrylamide (NIPA), and 3-acrylamidophenylboronic acid (AAPBA). The OCP PC has high stability and periodically-ordered structure, showing the desired structural color. The proposed PC material displays a red shift and reduced reflection intensity when detecting glucose molecules. The red-shift wavelength reaches 75 nm, which clearly changes the structural color from brilliant blue to emerald green. This visually distinguishable color change facilitates the detection of the glucose concentrations from 3 to 20 mm, which demonstrates the potential of the opal PC material for naked-eye detection. Thus, the novel PMMA–NIPA–AAPBA OCP PC is a simply prepared and sensitive material, which shows promising use in the diagnosis of diabetes mellitus and in real-time monitoring of diabetes. Different types of appropriated recognition groups are expected to be introduced into the 3D OCP PC to form new functional materials or chemical sensors, which will extensively broaden the PC material application. PMID:24829962

  18. Micrometer-level naked-eye detection of caesium particulates in the solid state

    NASA Astrophysics Data System (ADS)

    Mori, Taizo; Akamatsu, Masaaki; Okamoto, Ken; Sumita, Masato; Tateyama, Yoshitaka; Sakai, Hideki; Hill, Jonathan P.; Abe, Masahiko; Ariga, Katsuhiko

    2013-02-01

    Large amounts of radioactive material were released from the Fukushima Daiichi nuclear plant in Japan, contaminating the local environment. During the early stages of such nuclear accidents, iodine I-131 (half-life 8.02 d) is usually detectable in the surrounding atmosphere and bodies of water. On the other hand, in the long-term, soil and water contamination by Cs-137, which has a half-life of 30.17 years, is a serious problem. In Japan, the government is planning and carrying out radioactive decontamination operations not only with public agencies but also non-governmental organizations, making radiation measurements within Japan. If caesium (also radiocaesium) could be detected by the naked eye then its environmental remediation would be facilitated. Supramolecular material approaches, such as host-guest chemistry, are useful in the design of high-resolution molecular sensors and can be used to convert molecular-recognition processes into optical signals. In this work, we have developed molecular materials (here, phenols) as an optical probe for caesium cation-containing particles with implementation based on simple spray-on reagents and a commonly available fluorescent lamp for naked-eye detection in the solid state. This chemical optical probe provides a higher spatial resolution than existing radioscopes and gamma-ray cameras.

  19. Label-free silver nanoparticles for the naked eye detection of entecavir

    NASA Astrophysics Data System (ADS)

    Gao, Mengmeng; Lin, Rui; Li, Lili; Jiang, Li; Ye, Baofen; He, Hua; Qiu, Lanlan

    A simple, rapid, field-portable colorimetric method for the detection of entecavir was proposed based on the color change caused by the aggregation of silver nanoparticles. Neutralization of the electrostatic repulsion from each silver nanoparticle resulted in the aggregation of AgNPs and a consequent color change of AgNPs from yellow to wine-red, which provided a platform for rapid and field-portable colorimetric detection of entecavir. The concentration of entecavir could be determined with naked eye or UV-vis spectrometer. The proposed method can be used to detect entecavir in human urine with a detection limit of 1.51 μg mL-1, within 25 min by naked eye observation without the aid of any advanced instrument or complex pretreatment. Results from UV-vis spectra showed that the absorption ratio was linear with the concentration of entecavir in the range of 5.04-25.2 μg mL-1 and 1.01-5.04 μg mL-1 with linear coefficients of 0.9907 and 0.9955, respectively. The selectivity of AgNPs detection system for entecavir is excellent comparing with other ions and analytes. Due to its rapid, visible color changes, and excellent selectivity, the AgNPs synthesized in this study are suitable to be applied to on-site screening of entecavir in human urine.

  20. Naked-eye fingerprinting of single nucleotide polymorphisms on psoriasis patients

    NASA Astrophysics Data System (ADS)

    Valentini, Paola; Marsella, Alessandra; Tarantino, Paolo; Mauro, Salvatore; Baglietto, Silvia; Congedo, Maurizio; Paolo Pompa, Pier

    2016-05-01

    We report a low-cost test, based on gold nanoparticles, for the colorimetric (naked-eye) fingerprinting of a panel of single nucleotide polymorphisms (SNPs), relevant for the personalized therapy of psoriasis. Such pharmacogenomic tests are not routinely performed on psoriasis patients, due to the high cost of standard technologies. We demonstrated high sensitivity and specificity of our colorimetric test by validating it on a cohort of 30 patients, through a double-blind comparison with two state-of-the-art instrumental techniques, namely reverse dot blotting and sequencing, finding 100% agreement. This test offers high parallelization capabilities and can be easily generalized to other SNPs of clinical relevance, finding broad utility in diagnostics and pharmacogenomics.We report a low-cost test, based on gold nanoparticles, for the colorimetric (naked-eye) fingerprinting of a panel of single nucleotide polymorphisms (SNPs), relevant for the personalized therapy of psoriasis. Such pharmacogenomic tests are not routinely performed on psoriasis patients, due to the high cost of standard technologies. We demonstrated high sensitivity and specificity of our colorimetric test by validating it on a cohort of 30 patients, through a double-blind comparison with two state-of-the-art instrumental techniques, namely reverse dot blotting and sequencing, finding 100% agreement. This test offers high parallelization capabilities and can be easily generalized to other SNPs of clinical relevance, finding broad utility in diagnostics and pharmacogenomics. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr02200f

  1. A novel platform to enable inhaled naked RNAi medicine for lung cancer.

    PubMed

    Fujita, Yu; Takeshita, Fumitaka; Mizutani, Takayuki; Ohgi, Tadaaki; Kuwano, Kazuyoshi; Ochiya, Takahiro

    2013-01-01

    Small interfering RNA (siRNA)-based therapeutics have been used in humans and offer distinct advantages over traditional therapies. However, previous investigations have shown that there are several technical obstacles that need to be overcome before routine clinical applications are used. Currently, we are launching a novel class of RNAi therapeutic agents (PnkRNA™, nkRNA) that show high resistance to degradation and are less immunogenic, less cytotoxic, and capable of efficient intracellular delivery. Here, we develop a novel platform to promote naked RNAi approaches administered through inhalation without sophisticated delivery technology in mice. Furthermore, a naked and unmodified novel RNAi agent, such as ribophorin II (RPN2-PnkRNA), which has been selected as a therapeutic target for lung cancer, resulted in efficient inhibition of tumor growth without any significant toxicity. Thus, this new technology using aerosol delivery could represent a safe, potentially RNAi-based strategy for clinical applications in lung cancer treatment without delivery vehicles. PMID:24270189

  2. Naked-eye quantitative aptamer-based assay on paper device.

    PubMed

    Zhang, Yun; Gao, Dong; Fan, Jinlong; Nie, Jinfang; Le, Shangwang; Zhu, Wenyuan; Yang, Jiani; Li, Jianping

    2016-04-15

    This work initially describes the design of low-cost, naked-eye quantitative aptamer-based assays by using microfluidic paper-based analytical device (μPAD). Two new detection motifs are proposed for quantitative μPAD measurement without using external electronic readers, which depend on the length of colored region in a strip-like μPAD and the number of colorless detection microzones in a multi-zone μPAD. The length measuring method is based on selective color change of paper from colorless to blue-black via formation of iodine-starch complex. The counting method is conducted on the basis of oxidation-reduction reaction between hydrogen peroxide and potassium permanganate. Their utility is well demonstrated with sensitive, specific detection of adenosine as a model analyte with the naked eye in buffer samples and undiluted human serum. These equipment-free quantitative methods proposed thus hold great potential for the development of more aptamer-based assays that are simple, cost-efficient, portable, and user-friendly for various point-of-care applications particularly in resource-constrained environments. PMID:26684676

  3. Phylogeny of novel naked Filose and Reticulose Cercozoa: Granofilosea cl. n. and Proteomyxidea revised.

    PubMed

    Bass, David; Chao, Ema E-Y; Nikolaev, Sergey; Yabuki, Akinori; Ishida, Ken-Ichiro; Berney, Cédric; Pakzad, Ursula; Wylezich, Claudia; Cavalier-Smith, Thomas

    2009-02-01

    Naked filose and reticulose protozoa were long lumped as proteomyxids or left outside higher groups. We cultivated eight naked filose or reticulose strains, did light microscopy, 18S rDNA sequencing and phylogeny (showing all are Cercozoa), and sequenced 80 environmental 18S-types. Filose species belong in subphylum Filosa and reticulose ones in subphylum Endomyxa, making proteomyxids polyphyletic. We therefore transfer the classically mainly reticulose Proteomyxidea to Endomyxa, removing evident filosans as new class Granofilosea (including Desmothoracida, Acinetactis and new heliomonad family Heliomorphidae (new genus Heliomorpha (=Dimorpha)). Five new species of Limnofila gen. n. (L. mylnikovi; L. anglica; L. longa; L. oxoniensis; L. borokensis, previously misidentified as Biomyxa (=Gymnophrys) cometa) form a large freshwater clade (new order Limnofilida). Mesofila limnetica gen., sp. n. and Nanofila marina gen., sp. n. group separately in Granofilosea (Cryptofilida ord. n.). In Endomyxa, a new genus of reticulose proteomyxids (Filoreta marina, F. japonica, F. turcica spp. n., F. (=Corallomyxa) tenera comb. n.) forms a clade (Reticulosida) related to Gromiidea/Ascetosporea. Platyreta germanica gen., sp. n. and Arachnula impatiens are related vampyrellids (Aconchulinida) within a large clade beside Phytomyxea. Biomyxidae and Rhizoplasmidae fam. n. remain incertae sedis within Proteomyxidea. Gymnophrydium and Borkovia are revised. The reticulose Corallomyxa are unlike Filoreta and possibly Amoebozoa, not Cercozoa. PMID:18952499

  4. A new rhodamine-based colorimetric chemosensor for naked-eye detection of Cu2 + in aqueous solution

    NASA Astrophysics Data System (ADS)

    Hu, Yang; Zhang, Jing; Lv, Yuan-Zheng; Huang, Xiao-Huan; Hu, Sheng-li

    2016-03-01

    A new colorimetric probe 1 based on rhodamine B lactam was developed for naked-eye detection of Cu2 +. The optical feature of 1 for Cu2 + was investigated by UV-vis absorption spectroscopy. Upon the addition of Cu2 +, the 1 displayed a distinct color change from colorless to pink, which can be directly detected by the naked eye. The stoichiometry of 1 to Cu2 + complex was found to be 1:1 and the naked-eye detection limit was determined as low as 2 μM. The results suggest that the probe 1 may provide a convenient method for visual detection of Cu2 + with high sensitivity.

  5. Quantum probes of timelike naked singularities in the weak field regime of f ( R) global monopole spacetime

    NASA Astrophysics Data System (ADS)

    Gurtug, O.; Halilsoy, M.; Mazharimousavi, S. Habib

    2014-01-01

    The formation of a naked singularity in f ( R) global monopole spacetime is considered in view of quantum mechanics. Quantum test fields obeying the Klein-Gordon, Dirac and Maxwell equations are used to probe the classical timelike naked singularity developed at r = 0. We prove that the spatial derivative operator of the fields fails to be essentially self-adjoint. As a result, the classical timelike naked singularity formed in f ( R) global monopole spacetime remains quantum mechanically singular when it is probed with quantum fields having different spin structures. Pitelli and Letelier ( Phys. Rev. D 80 (2009) 104035) had shown that for quantum scalar ( spin 0) probes the general relativistic global monopole singularity remains intact. For specific modes electromagnetic ( spin 1) and Dirac field ( spin 1 /2) probes, however, we show that the global monopole spacetime behaves quantum mechanically regular. The admissibility of this singularity is also incorporated within the Gubser's singularity conjecture.

  6. Immunogenicity and Effectiveness of Routine Immunization With 1 or 2 Doses of Inactivated Poliovirus Vaccine: Systematic Review and Meta-analysis

    PubMed Central

    Grassly, Nicholas C.

    2014-01-01

    Background. The World Health Organization has recommended that all 124 countries currently using only oral poliovirus vaccine (OPV) introduce at least 1 dose of inactivated poliovirus vaccine (IPV) before the global withdrawal of serotype 2 OPV in 2016. A 1- or 2-dose schedule, potentially administered intradermally with reduced antigen content, may make this affordable. Methods. A systematic review and meta-analysis of studies documenting seroconversion after 1 or 2, full or fractional (1/5) doses of enhanced-potency IPV was performed. Studies reporting the clinical efficacy of IPV were also reviewed. Results. Twenty study arms from 12 published articles were included in the analysis of seroconversion. One full dose of intramuscular IPV seroconverted 33%, 41%, and 47% of infants against serotypes 1, 2, and 3 on average, whereas 2 full doses seroconverted 79%, 80%, and 90%, respectively. Seroconversion increased with age at administration. Limited data from case-control studies indicate clinical efficacy equivalent to the proportion seroconverting. One fractional dose of intradermal IPV gave lower seroconversion (10%–40%), but after 2 doses seroconversion was comparable to that with full-dose IPV. Conclusions. Routine immunization with 2 full or fractional doses of IPV given after 10 weeks of age is likely to protect >80% of recipients against poliomyelitis if poliovirus reemerges after withdrawal of OPV serotypes. PMID:24634499

  7. An Outbreak of Type Π Vaccine-Derived Poliovirus in Sichuan Province, China: Emergence and Circulation in an Under-Immunized Population

    PubMed Central

    Fan, Chun-Xiang; Liu, Qing-Lian; Hao, Li-Xin; Liu, Yu; Zheng, Jing-Shan; Qin, Zhi-Ying; Xia, Wei; Zhang, Shi-Yue; Yin, Zun-Dong; Jing, Qiong; Zhang, Yan-Xia; Huang, Rong-Na; Yang, Ru-Pei; Tong, Wen-Bin; Qi, Qi; Guan, Xu-Jing; Jing, Yu-Lin; Ma, Qian-Li; Wang, Jin; Ma, Xiao-Zhen; Chen, Na; Zheng, Hong-Ru; Li, Yin-Qiao; Ma, Chao; Su, Qi-Ru; Reilly, Kathleen H.; Luo, Hui-Ming; Wu, Xian-Ping; Wen, Ning; Yang, Wei-Zhong

    2014-01-01

    Background During August 2011–February 2012, an outbreak of type Π circulating vaccine-derived poliovirus (cVDPVs) occurred in Sichuan Province, China. Methods A field investigation of the outbreak was conducted to characterize outbreak isolates and to guide emergency response. Sequence analysis of poliovirus capsid protein VP1 was performed to determine the viral propagation, and a coverage survey was carried out for risk assessment. Results One clinical compatible polio case and three VDPV cases were determined in Ngawa County, Ngawa Tibetan and Qiang Autonomous Prefecture, Sichuan Province. Case patients were unimmunized children, 0.8–1 years old. Genetic sequencing showed that the isolates diverged from the VP1 region of the type Π Sabin strain by 5–12 nucleotides (nt) and shared the same 5 nt VP1 substitutions, which indicate single lineage of cVDPVs. Of the 7 acute flaccid paralysis cases (all>6 months) reported in Ngawa Prefecture in 2011, 4 (57.1%) cases (including 2 polio cases) did not receive oral attenuated poliovirus vaccine. Supplementary immunization activities (SIAs) were conducted in February–May, 2012, and the strain has not been isolated since. Conclusion High coverage of routine immunization should be maintained among children until WPV transmission is globally eradicated. Risk assessments should be conducted regularly to pinpoint high risk areas or subpopulations, with SIAs developed if necessary. PMID:25503964

  8. Translation of poliovirus RNA: role of an essential cis-acting oligopyrimidine element within the 5' nontranslated region and involvement of a cellular 57-kilodalton protein.

    PubMed Central

    Pestova, T V; Hellen, C U; Wimmer, E

    1991-01-01

    Translation of poliovirus RNA is initiated by cap-independent internal entry of ribosomes into the 5' nontranslated region. This process is dependent on elements within the 5' nontranslated region (the internal ribosomal entry site) and may involve novel translation factors. Systematic mutation of a conserved oligopyrimidine tract has revealed a cis-acting element that is essential for translation in vitro. The function of this element is related to its position relative to other cis-acting domains. This element is part of a more complex structure that interacts with several cellular factors, but changes in protein binding after mutation of this element were not detected in a UV cross-linking assay. A 57-kDa protein from the ribosomal salt wash fraction of HeLa cells was identified that binds upstream of the oligopyrimidine tract. Translation of poliovirus mRNA in vitro was strongly and specifically inhibited by competition with the p57-binding domain (nucleotides 260 to 488) of the 5' nontranslated region of encephalomyocarditis virus, indicating a probable role for p57 in poliovirus translation. p57 is likely to be identical to the ribosome-associated factor that binds to and is necessary for the function of the internal ribosomal entry site of encephalomyocarditis virus RNA. Images PMID:1656091

  9. Self-replicating Replicon-RNA Delivery to Dendritic Cells by Chitosan-nanoparticles for Translation In Vitro and In Vivo

    PubMed Central

    McCullough, Kenneth C; Bassi, Isabelle; Milona, Panagiota; Suter, Rolf; Thomann-Harwood, Lisa; Englezou, Pavlos; Démoulins, Thomas; Ruggli, Nicolas

    2014-01-01

    Self-amplifying replicon RNA (RepRNA) possesses high potential for increasing antigen load within dendritic cells (DCs). The major aim of the present work was to define how RepRNA delivered by biodegradable, chitosan-based nanoparticulate delivery vehicles (nanogel-alginate (NGA)) interacts with DCs, and whether this could lead to translation of the RepRNA in the DCs. Although studies employed virus replicon particles (VRPs), there are no reports on biodegradable, nanoparticulate vehicle delivery of RepRNA. VRP studies employed cytopathogenic agents, contrary to DC requirements—slow processing and antigen retention. We employed noncytopathogenic RepRNA with NGA, demonstrating for the first time the efficiency of RepRNA association with nanoparticles, NGA delivery to DCs, and RepRNA internalization by DCs. RepRNA accumulated in vesicular structures, with patterns typifying cytosolic release. This promoted RepRNA translation, in vitro and in vivo. Delivery and translation were RepRNA concentration-dependent, occurring in a kinetic manner. Including cationic lipids with chitosan during nanoparticle formation enhanced delivery and translation kinetics, but was not required for translation of immunogenic levels in vivo. This work describes for the first time the characteristics associated with chitosan-nanoparticle delivery of self-amplifying RepRNA to DCs, leading to translation of encoded foreign genes, namely influenza virus hemagglutinin and nucleoprotein. PMID:25004099

  10. Apple scar skin viroid naked RNA is actively transmitted by the whitefly Trialeurodes vaporariorum.

    PubMed

    Walia, Yashika; Dhir, Sunny; Zaidi, Aijaz Asghar; Hallan, Vipin

    2015-01-01

    Nucleic acid transfer between plants is a phenomenon which is likely to occur in many ways in nature. We report here the active transmission of Apple scar skin viroid (ASSVd) naked ssRNA species by the whitefly Trialeurodes vaporariorum (Tv). Not only the viroid RNA, its DNA form was also identified from the insect. The viroid transfer efficiency was enhanced with the help of Cucumis sativus Phloem protein 2 (CsPP2), a plant protein known to translocate viroid RNAs. This PP2/ASSVd complex is stably present in the viroid infected cucumber plants, as was identified with the help of immunological reaction. As viroid-like secondary structures are found in some plant RNAs, and PP2 is known to bind and translocate several RNAs, the results have huge implications in transfer of these RNA species between plants visited by the whitefly. PMID:26327493

  11. Analytic derivation of the map of null rays passing near a naked singularity

    SciTech Connect

    Tanaka, Takahiro; Singh, T. P.

    2001-06-15

    Recently the energy emission from a naked singularity forming in spherical dust collapse has been investigated. This radiation is due to particle creation in a curved spacetime. In this discussion, the central role is played by the mapping formula between the incoming and the outgoing null coordinates. For the self-similar model, this mapping formula has been derived analytically. But for the model with C{sup {infinity}} density profile, the mapping formula has been obtained only numerically. In the present paper, we argue that the singular nature of the mapping is determined by the local geometry around the point at which the singularity is first formed. If this is the case, it would be natural to expect that the mapping formula can be derived analytically. In the present paper, we analytically rederive the same mapping formula for the model with C{sup {infinity}} density profile that has been earlier derived using a numerical technique.

  12. Formation of naked singularities in five-dimensional space-time

    SciTech Connect

    Yamada, Yuta; Shinkai, Hisa-aki

    2011-03-15

    We numerically investigate the gravitational collapse of collisionless particles in spheroidal configurations both in four- and five-dimensional (5D) space-time. We repeat the simulation performed by Shapiro and Teukolsky (1991) that announced an appearance of a naked singularity, and also find similar results in the 5D version. That is, in a collapse of a highly prolate spindle, the Kretschmann invariant blows up outside the matter and no apparent horizon forms. We also find that the collapses in 5D proceed more rapidly than in 4D, and the critical prolateness for the appearance of an apparent horizon in 5D is loosened, compared to 4D cases. We also show how collapses differ with spatial symmetries comparing 5D evolutions in single-axisymmetry, SO(3), and those in double-axisymmetry, U(1)xU(1).

  13. Searching for the Thermal Plasma in the Naked Pulsar Wind Nebula CTB 87

    NASA Astrophysics Data System (ADS)

    Safi-Harb, Samar

    2013-10-01

    The missing thermal X-ray emission from dozens of naked pulsar wind nebulae (PWNe) remains one of the most puzzling questions in the SNR field. Our understanding of PWNe has been largely biased by the study of the brightest and youngest objects, such as the Crab nebula. We propose a deep EPIC observation of CTB 87. Our Chandra study, complemented with multi-wavelength observations, suggests that this remnant is an evolved PWN, most likely crushed by the supernova explosion's reverse shock. Alternatively, a bow-shock interpretation is also plausible. Our proposed observation will discriminate between these two scenarios. XMM-Newton is the ideal facility to perform a deep search for the thermal emission expected at the PWN-reverse shock interaction site and from the long-sought SNR shell.

  14. Hyaluronan in cancer - from the naked mole rat to nanoparticle therapy.

    PubMed

    Rankin, Kenneth S; Frankel, Daniel

    2016-05-01

    Hyaluronan, a glycosaminoglycan, abundant in the tumour microenvironment, is a key player in many processes associated with cancer. Recently the cancer resistance of the naked mole rat has been attributed to the presence of an ultra-high molecular weight form of this molecule. The physical properties of this multifunctional biopolymer have been extensively studied in the context of synovial joints. However, relatively little has been reported with regard to the soft matter properties of hyaluronan in relation to cancer. In this review we examine the role of hyaluronan in cancer, paying particular attention to its mechanical interactions with malignant cells and its soft matter properties. In addition we discuss the use of hyaluronan based gels to study cancer invasion as well as nanoparticle based strategies for disease treatment. PMID:27079782

  15. Self-Folding of Naked Segment 8 Genomic RNA of Influenza A Virus

    PubMed Central

    Lenartowicz, Elzbieta; Kesy, Julita; Ruszkowska, Agnieszka; Soszynska-Jozwiak, Marta; Michalak, Paula; Moss, Walter N.; Turner, Douglas H.; Kierzek, Ryszard; Kierzek, Elzbieta

    2016-01-01

    Influenza A is a negative sense RNA virus that kills hundreds of thousands of humans each year. Base pairing in RNA is very favorable, but possibilities for RNA secondary structure of the influenza genomic RNA have not been investigated. This work presents the first experimentally-derived exploration of potential secondary structure in an influenza A naked (protein-free) genomic segment. Favorable folding regions are revealed by in vitro chemical structure mapping, thermodynamics, bioinformatics, and binding to isoenergetic microarrays of an entire natural sequence of the 875 nt segment 8 vRNA and of a smaller fragment. Segment 8 has thermodynamically stable and evolutionarily conserved RNA structure and encodes essential viral proteins NEP and NS1. This suggests that vRNA self-folding may generate helixes and loops that are important at one or more stages of the influenza life cycle. PMID:26848969

  16. Naked eye determination of the dawn for Sinai and Assiut of Egypt

    NASA Astrophysics Data System (ADS)

    Hassan, A. H.; Issa, I. A.; Mousa, M.; Abdel-Hadi, Yasser A.

    2016-06-01

    Twilight observations were carried out by naked eye in the period (2010-2012) for north Sinai (Lat. 31°4‧N, Long. 32°52‧E) where the background is desert, and for Assiut (Lat. 27°10‧N, Long. 31°10‧E) in the period (2012-2014) where the background is agricultural land. The purpose of these observations is to calculate the depression of the sun below the horizon at which the normal eye can discriminate the dawn (morning white thread) for two sites. The results indicated that this discrimination takes place at vertical sun depression angles, Do = 14.61° and 13.665° at Sinai and Assiut respectively.

  17. Magnetic purification of curcumin from Curcuma longa rhizome by novel naked maghemite nanoparticles.

    PubMed

    Magro, Massimiliano; Campos, Rene; Baratella, Davide; Ferreira, Maria Izabela; Bonaiuto, Emanuela; Corraducci, Vittorino; Uliana, Maíra Rodrigues; Lima, Giuseppina Pace Pereira; Santagata, Silvia; Sambo, Paolo; Vianello, Fabio

    2015-01-28

    Naked maghemite nanoparticles, namely, surface active maghemite nanoparticles (SAMNs), characterized by a diameter of about 10 nm, possessing peculiar colloidal stability, surface chemistry, and superparamagnetism, present fundamental requisites for the development of effective magnetic purification processes for biomolecules in complex matrices. Polyphenolic molecules presenting functionalities with different proclivities toward iron chelation were studied as probes for testing SAMN suitability for magnetic purification. Thus, the binding efficiency and reversibility on SAMNs of phenolic compounds of interest in the pharmaceutical and food industries, namely, catechin, tyrosine, hydroxytyrosine, ferulic acid, coumaric acid, rosmarinic acid, naringenin, curcumin, and cyanidin-3-glucoside, were evaluated. Curcumin emerged as an elective compound, suitable for magnetic purification by SAMNs from complex matrices. A combination of curcumin, demethoxycurcumin, and bis-demethoxycurcumin was recovered by a single magnetic purification step from extracts of Curcuma longa rhizomes, with a purity >98% and a purification yield of 45%, curcumin being >80% of the total purified curcuminoids. PMID:25584520

  18. Naked eye observations for morning twilight at different sites in Egypt

    NASA Astrophysics Data System (ADS)

    Hassan, A. H.; Abdel-Hadi, Yasser A.; Issa, I. A.; Hassanin, N. Y.

    2014-06-01

    Twilight observations were carried out in the period between 1984 and 1987 in different seasons at different sites of Egypt (Baharia, Matrouh, Kottamia and Aswan) through a cooperation project between Dar El-Iftaa’ and the Egyptian Academy of Scientific Research and Technology. Naked eye observations of the first light of the dawn were done in parallel to the photoelectric measurements of the twilight phenomena. The depression of the sun below the horizon corresponding to the first light was calculated from the time of observations. Our estimates show that the normal eye can just discriminate the dawn (the first white light thread) at a depression of 14.7° with a maximum value of 15.08° and a minimum value of 12.01°. This result agrees with result obtained by our previous photoelectric measurements.

  19. X-ray sources in regions of star formation. I - The naked T Tauri stars

    NASA Technical Reports Server (NTRS)

    Walter, F. M.

    1986-01-01

    Einstein X-ray observations of regions of active star formation in Taurus, Ophiuchus, and Corona Australis show a greatly enhanced surface density of stellar X-ray sources over that seen in other parts of the sky. Many of the X-ray sources are identified with low-mass, pre-main-sequence stars which are not classical T Tauri stars. The X-ray, photometric, and spectroscopic data for these stars are discussed. Seven early K stars in Oph and CrA are likely to be 1-solar-mass post-T Tauri stars with ages of 10-million yr. The late K stars in Taurus are not post-T Tauri, but 'naked' T Tauri stars, which are coeval with the T Tauri stars, differing mainly in the lack of a circumstellar envelope.

  20. Naked-eye detection of biologically important anions by a new chromogenic azo-azomethine sensor

    NASA Astrophysics Data System (ADS)

    Rezaeian, Khatereh; Khanmohammadi, Hamid

    2014-12-01

    A new chromogenic azo-azomethine sensor, containing active phenolic sites, has been designed and synthesized via condensation reaction of N,N,N‧,N‧-tetrakis(2-aminoethyl)-2,2-dimethyl propane-1,3-diamine with 1-(3-formyl-4-hydroxyphenylazo)-4-nitrobenzene. The anion recognition ability of the synthesized receptor was evaluated using UV-Vis spectroscopy and 1H NMR technique. The anion recognition studies exhibited that the receptor acts as a sensor for biologically important anions such as F-, AcO- and H2PO4- over other anions. The binding stoichiometry between sensor and anions was found to be 1:2. 1H NMR experiment revealed that sensor recognizes anions via H-bonds and subsequent deprotonation to elicit a vivid color change. Interestingly, the sensory system not only let for the naked eye detection without any spectroscopic instrumentation but also helped to discriminate between anions.

  1. Naked-eye fingerprinting of single nucleotide polymorphisms on psoriasis patients.

    PubMed

    Valentini, Paola; Marsella, Alessandra; Tarantino, Paolo; Mauro, Salvatore; Baglietto, Silvia; Congedo, Maurizio; Paolo Pompa, Pier

    2016-06-01

    We report a low-cost test, based on gold nanoparticles, for the colorimetric (naked-eye) fingerprinting of a panel of single nucleotide polymorphisms (SNPs), relevant for the personalized therapy of psoriasis. Such pharmacogenomic tests are not routinely performed on psoriasis patients, due to the high cost of standard technologies. We demonstrated high sensitivity and specificity of our colorimetric test by validating it on a cohort of 30 patients, through a double-blind comparison with two state-of-the-art instrumental techniques, namely reverse dot blotting and sequencing, finding 100% agreement. This test offers high parallelization capabilities and can be easily generalized to other SNPs of clinical relevance, finding broad utility in diagnostics and pharmacogenomics. PMID:27174795

  2. Event monitoring of herbicides with naked and membrane-covered Empore disk integrative passive sampling devices.

    PubMed

    Stephens, B Scott; Kapernick, Anita P; Eaglesham, Geoff; Mueller, Jochen F

    2009-08-01

    Subsequent to an initial wet season flood event in the Brisbane River, Australia, both fast (naked disk) and slow (membrane-covered) variants of SDB-RPS Empore disk passive sampling devices were deployed with an automated grab sampling program. A trend increase in the aquatic dissolved concentrations of diuron and simazine was observed over a 10-day period. Kinetic and equilibrium parameters for each sampler were calculated based on the dynamic concentration. Absolute percent difference for duplicate passive samples was <10% in the fast and <25% in the slow samplers. For kinetic sampling, significantly shortened integrative periods are available with the fast compared with the slow variant, with higher sampling rates offering improved detection limits. The study demonstrates a method for determining kinetic parameters of passive samplers in a variable concentration field deployment, and illustrates the differences in quality between active and passive data, in terms of capturing changes in concentration associated with rainfall events. PMID:19520390

  3. Data on macrophage mediated muscle transfection upon delivery of naked plasmid DNA with block copolymers

    PubMed Central

    Mahajan, Vivek; Gaymalov, Zagit; Alakhova, Daria; Gupta, Richa; Zucker, Irving H.; Kabanov, Alexander V.

    2016-01-01

    The data contains 14 figures supporting the research article “Horizontal gene transfer from macrophages to ischemic muscles upon delivery of naked DNA with Pluronic block copolymers” [1]. The data explains the surgical procedure and histological characterization of Murine Hind Limb Ischemia. The data also shows the kinetics of luciferase gene expression, spread of GFP expression through muscle and the colocalization of GFP with cellular markers in ischemic muscles injected with pDNA alone or pDNA/Pluronic. Finally the data shows the effect of Pluronic Block Copolymer to enhance total gene expression (cmv-promoter driven luciferase gene) in coculture of DNA transfected MØs with muscle cells. PMID:27222845

  4. A sterically demanding organo-superbase avoids decomposition of a naked trifluoromethyl carbanion directly generated from fluoroform.

    PubMed

    Kawai, Hiroyuki; Yuan, Zhe; Tokunaga, Etsuko; Shibata, Norio

    2013-03-01

    A simple strategy avoiding the decomposition of a naked trifluoromethyl anion to difluorocarbene by a sterically very demanding organo-superbase without the help of a trifluoromethyl anion reservoir such as DMF is reported. The direct non-metallic trifluoromethylation of carbonyl compounds using fluoroform in the presence of t-Bu-P4 base afforded trifluoromethyl alcohols in high yields. PMID:23344691

  5. The effect of phytohormones on the dynamics of protein biosynthesis and enzyme activity in linted and naked cotton seed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We determined the effect of exogenous indole-3-acetic acid, a-naphthylene-3-acetic acid and gibberellic acid (GA3) on the enzymatic activity of glucansynthase, peroxidase and cellulase in ovule development of naked L-70 and linted AN-Bayaut-2 cotton (Gossypium hirsutum L.) seeds. We isolated a prote...

  6. Light-induced generation of singlet oxygen by naked gold nanoparticles and its implications to cancer cell phototherapy.

    PubMed

    Pasparakis, George

    2013-12-20

    Generation of singlet oxygen by direct irradiation of naked gold nanoparticles is observed using either continuous wave or pulsed laser sources. The underlying mechanism involves plasmon- and hot-electron-mediated reaction pathways and (1) O2 seems to significantly amplify the overall death rates during photothermal treatment of cancer cell lines in vitro. PMID:23813944

  7. Naked-eye detection as a universal approach to lower the limit of detection of enzyme-linked immunoassays.

    PubMed

    O'Connor, Erin F; Paterson, Sureyya; de la Rica, Roberto

    2016-05-01

    Colorimetric biosensors for the detection of analytes with the naked eye are required in environmental monitoring, point-of-care diagnostics, and analyses in resources constrained settings, where detection instruments may not be available. However, instrument-based detection methods are usually more adequate for detecting small variations in the signal compared to naked-eye detection schemes, and consequently the limit of detection of the latter is usually higher than the former. Here, we demonstrate that the limit of detection of colorimetric enzyme-linked immunoassays can be decreased several orders of magnitude when using naked-eye detection instead of a spectrophotometer for detecting the signal. The key step to lower the limit of detection is adding a small volume of chromogenic substrate during the signal generation step. This generates highly colored solutions that can be easily visualized with the naked eye and recorded with the camera of a mobile phone. The proposed method does not require expensive equipment or complex protocols to enhance the signal, and therefore it is a universal approach to lower the limit of detection of colorimetric enzyme-linked immunoassays. PMID:26970749

  8. Fast detection of oxygen by the naked eye using a stable metal-organic framework containing methyl viologen cations.

    PubMed

    Gong, Yun-Nan; Lu, Tong-Bu

    2013-09-11

    A stable porous metal-organic framework (MOF) containing methyl viologen cations exhibits reversible photochromic, thermochromic and fluorescence changes via host-guest interactions, and can be used for fast and selective detection of oxygen by naked eye recognition of color change within five seconds. PMID:23877538

  9. A cytosolic protein factor from the naked mole-rat activates proteasomes of other species and protects these from inhibition

    PubMed Central

    Rodriguez, Karl A.; Osmulski, Pawel A.; Pierce, Anson; Weintraub, Susan T.; Gaczynska, Maria; Buffenstein, Rochelle

    2015-01-01

    The naked mole-rat maintains robust proteostasis and high levels of proteasome-mediated proteolysis for most of its exceptional (~31y) life span. Here, we report that the highly active proteasome from the naked mole-rat liver resists attenuation by a diverse suite of proteasome-specific small molecule inhibitors. Moreover, mouse, human, and yeast proteasomes exposed to the proteasome-depleted, naked mole-rat cytosolic fractions, recapitulate the observed inhibition resistance, and mammalian proteasomes also show increased activity. Gel filtration coupled with mass spectrometry and atomic force microscopy indicates that these traits are supported by a protein factor that resides in the cytosol. This factor interacts with the proteasome and modulates its activity. Although HSP72 and HSP40 (Hdj1) are among the constituents of this factor, the observed phenomenon, such as increasing peptidase activity and protecting against inhibition cannot be reconciled with any known chaperone functions. This novel function may contribute to the exceptional protein homeostasis in the naked mole-rat and allow it to successfully defy aging. PMID:25018089

  10. Dreams and Reality of Using Naked Ge Detectors in Liquid Nitrogen Status (Long-Term Stability) of GENIUS-TF

    NASA Astrophysics Data System (ADS)

    Krivosheina, Irina V.; Klapdor-Kleingrothaus, Hans V.

    2008-04-01

    GENIUS-TF-II is a setup of six naked high purity Ge detectors (15 kg) in liquid nitrogen in Gran Sasso. It has been installed in October, 2004 - after the first four naked Ge detectors had been installed on May 5, 2003 (GENIUS-TF-I). The GENIUS-Test-Facility (GENIUS-TF) is the first and up to now only setup ever testing the novel technique aiming at extreme background reduction in search for rare decays in particular underground. The goal of GENIUS-TF was to test some key operational parameters of the full GENIUS project in 1997.1-6 Simultaneous physical goal was to search for the annual modulation of the Dark Matter signal.12,25 After operation of GENIUS-TF over three years with finally six naked Ge detectors (15 kg) in liquid nitrogen in Gran Sasso we realize serious problems for realization of a full-size GENIUS-like experiment: 1. Background from 222Rn diffusing into the setup, on a level far beyond the expectation. 2. Limited long-term stability of naked detectors in liquid nitrogen as result of increasing leakage current. None of the six detectors is running after three years with the nominal leakage current. Three of the six detectors do not work any more at all. The results of our three years of investigation of the long-term stability casts serious doubt on the possibility to perform a full GENIUS project - or its copies GERDA or CAMEO/GEM.

  11. Phage-mediated counting by the naked eye of miRNA molecules at attomolar concentrations in a Petri dish

    NASA Astrophysics Data System (ADS)

    Zhou, Xin; Cao, Peng; Zhu, Ye; Lu, Wuguang; Gu, Ning; Mao, Chuanbin

    2015-10-01

    The ability to count biomolecules such as cancer-biomarker miRNAs with the naked eye is seemingly impossible in molecular diagnostics. Here, we show an ultrasensitive naked-eye-counting strategy for quantifying miRNAs by employing T7 phage--a bacteria-specific virus nanoparticle--as a surrogate. The phage is genetically engineered to become fluorescent and capable of binding a miRNA-capturing gold nanoparticle (GNP) in a one-to-one manner. Target miRNAs crosslink the resultant phage-GNP couple and miRNA-capturing magnetic microparticles, forming a sandwich complex containing equimolar phage and miRNA. The phage is then released from the complex and developed into one macroscopic fluorescent plaque in a Petri dish by plating it in a host bacterial medium. Counting the plaques by the naked eye enables the quantification of miRNAs with detection limits of ~3 and ~5 aM for single-target and two-target miRNAs, respectively. This approach offers ultrasensitive and convenient quantification of disease biomarkers by the naked eye.

  12. Social and Hormonal Triggers of Neural Plasticity in Naked Mole-Rats

    PubMed Central

    Holmes, Melissa M.; Seney, Marianne L.; Goldman, Bruce D.; Forger, Nancy G.

    2010-01-01

    Naked mole-rats are eusocial rodents that live in large social groups with a strict reproductive hierarchy. In each colony only a few individuals breed; all others are non-reproductive subordinates. We previously showed that breeders have increased volume of several brain regions linked to reproduction: the paraventricular nucleus of the hypothalamus (PVN), the principal nucleus of the bed nucleus of the stria terminalis (BSTp), and the medial amygdala (MeA). Breeders also have more large motoneurons in Onuf’s nucleus (ON) in the spinal cord, a cell group innervating perineal muscles that attach to the genitalia. Here, we sought to determine triggers for the neural changes seen in breeders. Specifically, we compared four groups of animals: subordinates, paired animals that did not reproduce, gonadally intact breeders, and gonadectomized breeders. We find that pairing alone is sufficient to cause breeder-like changes in volume of the PVN and cell size distribution in ON. In contrast, increases in BSTp volume were seen only in animals that actually reproduced. Those changes that were seen in successful breeders appear to be independent of gonadal steroids because long-term gonadectomy did not reverse the breeder-like neural changes in the PVN, BSTp or ON, although a trend for gonadectomized animals having larger MeA volumes was detected. Thus, neural changes associated with breeding status in naked mole-rats may be triggered by different aspects of the social and reproductive environment; once changes occur they are largely independent of gonadal hormones and may be permanent. PMID:21130812

  13. Prevalence of Acanthamoeba and other naked amoebae in South Florida domestic water.

    PubMed

    Shoff, M E; Rogerson, A; Kessler, K; Schatz, S; Seal, D V

    2008-03-01

    The purpose was to identify the prevalence of naked amoebae in tap water in south Florida to ascertain the risk of amoebal infections of the cornea in contact lens wearers. Over the course of a 2-year period, water samples were collected from sites throughout Broward, Palm Beach, and Dade counties, Florida. The presence of amoebae in samples was based on an enrichment cultivation method appropriate for Acanthamoeba. Amoebae were identified using diagnostic features discernable by light microscopy. A total of 283 water samples were processed and amoebae were noted in 80 of these. Acanthamoeba were found on 8 occasions (2.8%). The genera Hartmannella and Vahlkampfia, rarely involved in keratitis cases, were found in 3.5% and 2.8% of samples, respectively. A total of 19 different naked amoebae were recorded and amoebae (regardless of genus) were present in 19.4% of all samples. Previous surveys in England and Korea have shown that acanthamoebae are found in 15 to 30% of tap water samples in the home and have been associated with corneal infection in contact lens wearers. The incidence of acanthamoebae infection in the USA (2.8%) has been found to be lower than that in the UK and it has been postulated that this is related to the lack of a storage water tank in the roof loft space. However, the level of treatment of municipal water is clearly not effective at killing amoebal cysts (or trophozoites) as evidenced by the high occurrence of amoebae (19.4%) in this study. PMID:17998610

  14. Contribution of Contact Sampling in Increasing Sensitivity of Poliovirus Detection During A Polio Outbreak-Somalia, 2013.

    PubMed

    Moturi, Edna; Mahmud, Abdirahman; Kamadjeu, Raoul; Mbaeyi, Chukwuma; Farag, Noha; Mulugeta, Abraham; Gary, Howard; Ehrhardt, Derek

    2016-04-01

    Background.  In May 2013, a wild poliovirus type 1 (WPV1) outbreak reported in Somalia provided an opportunity to examine the contribution of testing contacts to WPV detection. Methods.  We reviewed acute flaccid paralysis (AFP) case-patients and linked contacts reported in the Somalia Surveillance Database from May 9 to December 31, 2013. We restricted our analysis to AFP case-patients that had ≥3 contacts and calculated the contribution of each contact to case detection. Results.  Among 546 AFP cases identified, 328 AFP cases had ≥3 contacts. Among the 328 AFP cases with ≥3 contacts, 93 WPV1 cases were detected: 58 cases (62%; 95% confidence interval [CI], 52%-72%) were detected through testing stool specimens from AFP case-patients; and 35 cases (38%; 95% CI, 28%-48%) were detected through testing stool specimens from contacts, including 19 cases (20%; 95% CI, 14%-30%) from the first contact, 11 cases (12%; 95% CI, 7%-20%) from the second contact, and 5 cases (5%; 95% CI, 2%-12%) from the third contact. Among the 103 AFP cases with ≥4 contacts, 3 (6%; 95% CI, 2%-16%) of 52 WPV1 cases were detected by testing the fourth contact. No additional WPV1 cases were detected by testing >4 contacts. Conclusions.  Stool specimens from 3 to 4 contacts of persons with AFP during polio outbreaks are needed to maximize detection of WPV cases. PMID:27419182

  15. Comparative in vivo efficiencies of hand-washing agents against hepatitis A virus (HM-175) and poliovirus type 1 (Sabin).

    PubMed Central

    Mbithi, J N; Springthorpe, V S; Sattar, S A

    1993-01-01

    The abilities of 10 hygienic hand-washing agents and tap water (containing approximately 0.5 ppm of free chlorine) to eliminate strain HM-175 of hepatitis A virus (HAV) and poliovirus (PV) type 1 (Sabin) were compared by using finger pad and whole-hand protocols with three adult volunteers. A mixture of the two viruses was prepared in a 10% suspension of feces, and 10 microliters of the mixture was placed on each finger pad. The inoculum was allowed to dry for 20 min, and the contaminated area was exposed to a hand-washing agent for 10 s, rinsed in tap water, and dried with a paper towel. In the whole-hand protocol, the hands were contaminated with 0.5 ml of the virus mixture, exposed for 10 s to a hand-washing agent, washed, and dried as described above. Tryptose phosphate broth was used to elute any virus remaining on the finger pads or hands. One part of the eluate was assayed directly for PV with FRhK-4 cells, while the other part was first treated with a PV-neutralizing serum and then assayed for HAV with the same cell line. The results are reported as mean percentages of reduction in PFU compared with the amount of infectious virus detectable after initial drying.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8250567

  16. Sero-Survey of Polio Antibodies during Wild Poliovirus Outbreak in Southern Xinjiang Uygur Autonomous Region, China

    PubMed Central

    Wang, Hai-Bo; Zhu, Shuang-Li; Zheng, Jing-Shan; Gou, Ai-Li; Cui, Hui; Zhang, Yong; Ning, Gui-Jun; Fan, Chun-Xiang; Chen, Yuan-Sheng; Li, Ke-Li; Yuan, Ping; Ma, Chao; Ma, Jing; Zheng, Hui; Fan, Xin-Chun; Li, Xin-Lan; Tang, Hai-Shu; Li, Xiao-Lei; Zhang, Fan; Yan, Dong-Mei; Wang, Dong-Yan; Cui, Zhi-Qiang; Ren, Li-Ping; Zhu, Hui; Wang, Hui-Ling; Jiang, Xiao-Hong; An, Hong-Qiu; Liu, Yang; Li, Jing; Xu, Wen-Bo; Wen, Ning; Xu, Ai-Qiang; Luo, Hui-Ming

    2014-01-01

    Background After being polio free for more than 10 years, an outbreak following importation of wild poliovirus (WPV) was confirmed in Xinjiang Uygur Autonomous Region, China, in 2011. Methods A cross-sectional study was conducted prior to supplementary immunization activities (SIAs), immediately after the confirmation of the WPV outbreak. In selected prefectures, participants aged ≤60 years old who visited hospitals at county-level or above to have their blood drawn for reasons not related to the study, were invited to participate in our study. Antibody titers ≥8 were considered positive. Results Among the 2,611 participants enrolled, 2,253 (86.3%), 2,283 (87.4%), and 1,989 (76.2%) were seropositive to P1, P2 and P3 respectively, and 1744 (66.8%) participants were seropositive to all the three serotypes. Lower antibody seropositivities and geometric mean titers were observed in children <1 year of age and in adults aged 15–39 years. Conclusion Serosurveys to estimate population immunity in districts at high risk of polio importation might be useful to gauge underlying population immunity gaps to polio and possibly to guide preparedness and response planning. Consideration should be given to older children and adults during polio risk assessment planning and outbreak response. PMID:24991811

  17. Contribution of Contact Sampling in Increasing Sensitivity of Poliovirus Detection During A Polio Outbreak—Somalia, 2013

    PubMed Central

    Moturi, Edna; Mahmud, Abdirahman; Kamadjeu, Raoul; Mbaeyi, Chukwuma; Farag, Noha; Mulugeta, Abraham; Gary, Howard; Ehrhardt, Derek

    2016-01-01

    Background. In May 2013, a wild poliovirus type 1 (WPV1) outbreak reported in Somalia provided an opportunity to examine the contribution of testing contacts to WPV detection. Methods. We reviewed acute flaccid paralysis (AFP) case-patients and linked contacts reported in the Somalia Surveillance Database from May 9 to December 31, 2013. We restricted our analysis to AFP case-patients that had ≥3 contacts and calculated the contribution of each contact to case detection. Results. Among 546 AFP cases identified, 328 AFP cases had ≥3 contacts. Among the 328 AFP cases with ≥3 contacts, 93 WPV1 cases were detected: 58 cases (62%; 95% confidence interval [CI], 52%–72%) were detected through testing stool specimens from AFP case-patients; and 35 cases (38%; 95% CI, 28%–48%) were detected through testing stool specimens from contacts, including 19 cases (20%; 95% CI, 14%–30%) from the first contact, 11 cases (12%; 95% CI, 7%–20%) from the second contact, and 5 cases (5%; 95% CI, 2%–12%) from the third contact. Among the 103 AFP cases with ≥4 contacts, 3 (6%; 95% CI, 2%–16%) of 52 WPV1 cases were detected by testing the fourth contact. No additional WPV1 cases were detected by testing >4 contacts. Conclusions. Stool specimens from 3 to 4 contacts of persons with AFP during polio outbreaks are needed to maximize detection of WPV cases. PMID:27419182

  18. Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase

    SciTech Connect

    Marcotte,L.; Wass, A.; Gohara, D.; Pathak, H.; Arnold, J.; Filman, D.; Cameron, C.; Hogle, J.

    2007-01-01

    Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3Cpro and the viral polymerase 3Dpol and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4- Angstroms resolution and the G64S fidelity mutant of 3Dpol at a 3.0- Angstroms resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3Dpol G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3Dpol makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.

  19. Modifications of both selectivity factor and upstream binding factor contribute to poliovirus-mediated inhibition of RNA polymerase I transcription.

    PubMed

    Banerjee, Rajeev; Weidman, Mary K; Navarro, Sonia; Comai, Lucio; Dasgupta, Asim

    2005-08-01

    Soon after infection, poliovirus (PV) shuts off host-cell transcription, which is catalysed by all three cellular RNA polymerases. rRNA constitutes more than 50 % of all cellular RNA and is transcribed from rDNA by RNA polymerase I (pol I). Here, evidence has been provided suggesting that both pol I transcription factors, SL-1 (selectivity factor) and UBF (upstream binding factor), are modified and inactivated in PV-infected cells. The viral protease 3C(pro) appeared to cleave the TATA-binding protein-associated factor 110 (TAF(110)), a subunit of the SL-1 complex, into four fragments in vitro. In vitro protease-cleavage assays using various mutants of TAF(110) and purified 3C(pro) indicated that the Q(265)G(266) and Q(805)G(806) sites were cleaved by 3C(pro). Both SL-1 and UBF were depleted in PV-infected cells and their disappearance correlated with pol I transcription inhibition. rRNA synthesis from a template containing a human pol I promoter demonstrated that both SL-1 and UBF were necessary to restore pol I transcription fully in PV-infected cell extracts. These results suggested that both SL-1 and UBF are transcriptionally inactivated in PV-infected HeLa cells. PMID:16033979

  20. Development of an efficient entire-capsid-coding-region amplification method for direct detection of poliovirus from stool extracts.

    PubMed

    Arita, Minetaro; Kilpatrick, David R; Nakamura, Tomofumi; Burns, Cara C; Bukbuk, David; Oderinde, Soji B; Oberste, M Steven; Kew, Olen M; Pallansch, Mark A; Shimizu, Hiroyuki

    2015-01-01

    Laboratory diagnosis has played a critical role in the Global Polio Eradication Initiative since 1988, by isolating and identifying poliovirus (PV) from stool specimens by using cell culture as a highly sensitive system to detect PV. In the present study, we aimed to develop a molecular method to detect PV directly from stool extracts, with a high efficiency comparable to that of cell culture. We developed a method to efficiently amplify the entire capsid coding region of human enteroviruses (EVs) including PV. cDNAs of the entire capsid coding region (3.9 kb) were obtained from as few as 50 copies of PV genomes. PV was detected from the cDNAs with an improved PV-specific real-time reverse transcription-PCR system and nucleotide sequence analysis of the VP1 coding region. For assay validation, we analyzed 84 stool extracts that were positive for PV in cell culture and detected PV genomes from 100% of the extracts (84/84 samples) with this method in combination with a PV-specific extraction method. PV could be detected in 2/4 stool extract samples that were negative for PV in cell culture. In PV-positive samples, EV species C viruses were also detected with high frequency (27% [23/86 samples]). This method would be useful for direct detection of PV from stool extracts without using cell culture. PMID:25339406

  1. The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons.

    PubMed

    Lahser, Frederick C; Bystol, Karin; Curry, Stephanie; McMonagle, Patricia; Xia, Ellen; Ingravallo, Paul; Chase, Robert; Liu, Rong; Black, Todd; Hazuda, Daria; Howe, Anita Y M; Asante-Appiah, Ernest

    2016-05-01

    The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for

  2. Transcriptome profiling analysis of naked carp (Gymnocypris przewalskii) provides insights into the immune-related genes in highland fish.

    PubMed

    Tong, Chao; Zhang, Cunfang; Zhang, Renyi; Zhao, Kai

    2015-10-01

    The naked carp, Gymnocypris przewalskii, is one of the dominant aquaculture fish species in Qinghai Province, China. Its wild stocks have severely suffered from overfishing, and the farming species are vulnerable to various pathogens infections. Here we report the first immune-related tissues transcriptome of a wild naked carp using a deep sequencing approach. A total of 158,087 unigenes are generated, 2687 gill-specific gene and 3215 kidney-specific genes are identified, respectively. Gene ontology analysis shows that 51,671 unigenes are involved in three major functional categories: biological process, cellular component, and molecular function. Further analysis shows that numerous consensus sequences are homologous to known immune-related genes. Pathways mapping annotate 56,270 unigenes and identify a large number of immune-related pathways. In addition, we focus on the immune-related genes and gene family in Toll-like receptor signaling pathway involved in innate immunity, including toll-like receptors (TLRs), interferon regulatory factors (IRFs), interleukins (ILs) and tumor necrosis factors (TNFs). Eventually, we identify 5 TLRs, 4 IRFs, 3 ILs and 2 TNFs with a completed coding sequence though mining the transcriptome data. Phylogeny analysis shows these genes of naked carp are mostly close to zebrafish. Protein domain and selection pressure analyses together show that all these genes are highly conserved in gene sequence and protein domain structure with other species, and purifying selection underwent in these genes, implied functionally important features are conserved in the genes above. Intriguingly, we detect positive selection signals in naked carp TLR4, and significant divergence occurred among tested species TLR4, suggested that naked carp TLR4 function may be affected. Finally, we identify 23,867 simple sequence repeat (SSR) marks in this transcriptome. Taken together, this study not only contributes a large number of candidate genes in naked carp

  3. The initial value problem for linearized gravitational perturbations of the Schwarzschild naked singularity

    NASA Astrophysics Data System (ADS)

    Dotti, Gustavo; Gleiser, Reinaldo J.

    2009-11-01

    The coupled equations for the scalar modes of the linearized Einstein equations around Schwarzschild's spacetime were reduced by Zerilli to a (1+1) wave equation \\partial ^2 \\Psi _z / \\partial t^2 + {\\cal H} \\Psi _z =0 , where {\\cal H} = -\\partial ^2 / \\partial x^2 + V(x) is the Zerilli 'Hamiltonian' and x is the tortoise radial coordinate. From its definition, for smooth metric perturbations the field Ψz is singular at rs = -6M/(ell - 1)(ell +2), with ell being the mode harmonic number. The equation Ψz obeys is also singular, since V has a second-order pole at rs. This is irrelevant to the black hole exterior stability problem, where r > 2M > 0, and rs < 0, but it introduces a non-trivial problem in the naked singular case where M < 0, then rs > 0, and the singularity appears in the relevant range of r (0 < r < ∞). We solve this problem by developing a new approach to the evolution of the even mode, based on a new gauge invariant function, \\hat{\\Psi} , that is a regular function of the metric perturbation for any value of M. The relation of \\hat{\\Psi} to Ψz is provided by an intertwiner operator. The spatial pieces of the (1 + 1) wave equations that \\hat{\\Psi} and Ψz obey are related as a supersymmetric pair of quantum Hamiltonians {\\cal H} and \\hat{\\cal H} . For M<0, \\hat{\\cal H} has a regular potential and a unique self-adjoint extension in a domain {\\cal D} defined by a physically motivated boundary condition at r = 0. This allows us to address the issue of evolution of gravitational perturbations in this non-globally hyperbolic background. This formulation is used to complete the proof of the linear instability of the Schwarzschild naked singularity, by showing that a previously found unstable mode belongs to a complete basis of \\hat{\\cal H} in {\\cal D} , and thus is excitable by generic initial data. This is further illustrated by numerically solving the linearized equations for suitably chosen initial data.

  4. Virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs.

    PubMed

    Eck, Melanie; Durán, Margarita García; Ricklin, Meret E; Locher, Samira; Sarraseca, Javier; Rodríguez, María José; McCullough, Kenneth C; Summerfield, Artur; Zimmer, Gert; Ruggli, Nicolas

    2016-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, were generated and evaluated as a potential vector vaccine against PRRSV infection. High level expression of the recombinant PRRSV proteins was demonstrated in cell culture. However, none of the PRRSV antigens expressed from VRP, with the exception of the N protein, did induce any detectable antibody response in pigs before challenge infection with PRRSV. After challenge however, the antibody responses against GP5, GP4 and GP3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control VRP. No reduction of viremia was observed in the vaccinated group compared with the control group. When pigs were co-vaccinated with VRP expressing IAV antigens and VRP expressing PRRSV glycoproteins, only antibody responses to the IAV antigens were detectable. These data show that the VSV replicon vector can induce immune responses to heterologous proteins in pigs, but that the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic. Nevertheless, they prime the immune system for significantly earlier B-cell responses following PRRSV challenge infection. PMID:26895704

  5. Diagnostic Potential and Antigenic Properties of Recombinant Tick-Borne Encephalitis Virus Subviral Particles Expressed in Mammalian Cells from Semliki Forest Virus Replicons

    PubMed Central

    Kuivanen, Suvi; Matveev, Andrey; Swaminathan, Sathyamangalam; Jääskeläinen-Hakala, Anu; Vapalahti, Olli

    2014-01-01

    The precursor membrane envelope (prME) proteins of all three tick-borne encephalitis virus (TBEV) subtypes were produced based on expression from Semliki Forest virus (SFV) replicons transcribed from recombinant plasmids. Vero E6 cells transfected by these plasmids showed specific reactivities in immunofluorescence and immunoblot assays by monoclonal antibodies against European and Far-Eastern subtype strains of TBEV, indicating proper folding of the expressed glycoproteins. The prME glycoproteins were secreted into the cell culture supernatant, forming TBEV subviral particles of 20 to 30 nm in diameter. IgM μ-capture and IgG monoclonal antibody (MAb)-capture enzyme immunoassays (EIAs) were developed based on prME Karelia-94 (Siberian subtype) particles. Altogether, 140 human serum samples were tested using these assays, and the results were compared to those obtained with a commercial IgM EIA, an in-house μ-capture IgM assay based on baculovirus-expressed antigen, a commercial IgG EIA, and a hemagglutination inhibition test. Compared to reference enzyme-linked immunosorbent assays (ELISAs), the sensitivities of the generated μ-capture IgM SFV-prME and IgG MAb-capture SFV-prME EIAs were 97.4 to 100% and 98.7%, respectively, and the specificities of the two assays were 100%. IgM and IgG immunofluorescence assays (IFAs) were created based on Vero E6 cells transfected with the recombinant plasmid carrying the TBEV Karelia-94 prME glycoproteins. The IgM IFA was 100% concordant with the μ-capture IgM bac-prME ELISA. The IgG IFA sensitivity and specificity were 98.7% and 100%, respectively, compared to those of the commercial ELISA. In conclusion, the tests developed based on SFV replicon-driven expression of TBEV glycoproteins provide safe and robust alternatives for conducting TBEV serology. PMID:24371235

  6. Single-Digit Pathogen and Attomolar Detection with the Naked Eye Using Liposome-Amplified Plasmonic Immunoassay.

    PubMed

    Bui, Minh-Phuong Ngoc; Ahmed, Snober; Abbas, Abdennour

    2015-09-01

    We introduce an enzyme-free plasmonic immunoassay with a binary (all-or-none) response. The presence of a single pathogen in the sample results in a chemical cascade reaction leading to a large red to dark-blue colorimetric shift visible to the naked eye. The immediate and amplified response is initiated by a triggered breakdown of cysteine-loaded nanoliposomes and subsequent aggregation of plasmonic gold nanoparticles. Our approach enabled visual detection of a single-digit live pathogen of Salmonella, Listeria, and E. coli O157 in water and food samples. Furthermore, the assay allowed a naked-eye detection of target antibody concentrations as low as 6.7 attomolar (600 molecules in 150 μL); six orders of magnitude lower than conventional enzyme-linked immunosorbent assay (ELISA). PMID:26308387

  7. GRB 080319B: A Naked-Eye Stellar Blast from the Distant Universe

    SciTech Connect

    Racusin, J. L.; Burrows, D. N.

    2008-10-22

    On behalf of a large international collaboration [1], we present the unprecedented broadband observations of GRB 080319B, whose prompt optical emission peaked at a visual magnitude of 5.3, making it briefly visible with the naked eye. GRB 080319B was discovered by Swift and captured in exquisite detail by ground based wide-field telescopes, imaging the burst location from before the time of the explosion. The combination of these unique optical data with simultaneous {gamma}-ray observations provides powerful diagnostics of the detailed physics of this explosion within seconds of its formation. We show that the prompt optical and {gamma}-ray emissions from this event arise from different spectral components within the same physical region located at a large distance from the source, implying an extremely relativistic outflow. Our observations also provide good evidence for a bright reverse shock component. The chromatic behavior of the broadband afterglow is consistent with viewing the GRB down the very narrow inner core of a two-component jet that is expanding into a wind-like environment consistent with the massive star origin of long GRBs.

  8. Self-similar cosmological solutions with dark energy. II. Black holes, naked singularities, and wormholes

    SciTech Connect

    Maeda, Hideki; Harada, Tomohiro; Carr, B. J.

    2008-01-15

    We use a combination of numerical and analytical methods, exploiting the equations derived in a preceding paper, to classify all spherically symmetric self-similar solutions which are asymptotically Friedmann at large distances and contain a perfect fluid with equation of state p=({gamma}-1){mu} with 0<{gamma}<2/3. The expansion of the Friedmann universe is accelerated in this case. We find a one-parameter family of self-similar solutions representing a black hole embedded in a Friedmann background. This suggests that, in contrast to the positive pressure case, black holes in a universe with dark energy can grow as fast as the Hubble horizon if they are not too large. There are also self-similar solutions which contain a central naked singularity with negative mass and solutions which represent a Friedmann universe connected to either another Friedmann universe or some other cosmological model. The latter are interpreted as self-similar cosmological white hole or wormhole solutions. The throats of these wormholes are defined as two-dimensional spheres with minimal area on a spacelike hypersurface and they are all nontraversable because of the absence of a past null infinity.

  9. Structural and optical properties of the naked and passivated Al5Au5 bimetallic nanoclusters

    NASA Astrophysics Data System (ADS)

    Grande-Aztatzi, Rafael; Formoso, Elena; Mercero, Jose M.; Matxain, Jon M.; Grabowski, Slawomir J.; Ugalde, Jesus M.

    2016-03-01

    The structural and optical properties of both the naked and passivated bimetallic Al5Au5 nanoclusters have been analyzed based on data obtained from ab initio density functional theory and quantum molecular dynamics simulations. It has been found that the Al5Au5 nanocluster possesses a hollow shaped minimum energy structure with segregated Al and Au layered domains, the former representing the electrophilic domain and the latter the nucleophilic domain. In particular, it has been shown that alkali metal cations attach in the nucleophilic domain and hop from one Au site to the next one in the picoseconds time scale, while anions are bound tightly to the Al atoms of the electrophilic domain. Simulating annealing studies are very suggestive of the proneness of the nanocluster towards coalescence into large cluster units, when the cluster is left unprotected by appropriate ligands. Further passivation studies with NaF salt suggest, nonetheless, the possibility of the isolation of the Al5Au5 cluster in molten salts or ionic liquids.

  10. ON THE RARITY OF X-RAY BINARIES WITH NAKED HELIUM DONORS

    SciTech Connect

    Linden, T.; Valsecchi, F.; Kalogera, V.

    2012-04-01

    The paucity of known high-mass X-ray binaries (HMXBs) with naked He donor stars (hereafter He star) in the Galaxy has been noted over the years as a surprising fact, given the significant number of Galactic HMXBs containing H-rich donors, which are expected to be their progenitors. This contrast has further sharpened in light of recent observations uncovering a preponderance of HMXBs hosting loosely bound Be donors orbiting neutron stars (NSs), which would be expected to naturally evolve into He-HMXBs through dynamical mass transfer onto the NS and a common-envelope (CE) phase. Hence, reconciling the large population of Be-HMXBs with the observation of only one He-HMXB can help constrain the dynamics of CE physics. Here, we use detailed stellar structure and evolution models and show that binary mergers of HMXBs during CE events must be common in order to resolve the tension between these observed populations. We find that, quantitatively, this scenario remains consistent with the typically adopted energy parameterization of CE evolution, yielding expected populations which are not at odds with current observations. However, future observations which better constrain the underlying population of loosely bound O/B-NS binaries are likely to place significant constraints on the efficiency of CE ejection.

  11. Surface-Induced Hydrogelation for Fluorescence and Naked-Eye Detections of Enzyme Activity in Blood.

    PubMed

    Xu, Tengyan; Liang, Chunhui; Ji, Shenglu; Ding, Dan; Kong, Deling; Wang, Ling; Yang, Zhimou

    2016-07-19

    Fluorescence probes have been widely applied for the detection of important analytes with high sensitivity and specificity. However, they cannot be directly applied for the detection in samples with autofluorescence such as blood. Herein, we demonstrated a simple but effective method of surface-induced self-assembly/hydrogelation for fluorescence detection of an enzyme in biological fluids including blood and cell lysates. The method utilizes an attracting glass surface to induce self-assembly of an enzyme-generating fluorescent probe. After removing the upper solution, the fluorescence turn-on at the glass surface can therefore be used for the detection of enzyme activity. By judging the thickness and color depth of hydrogels at the surface of the glass plates, we could also estimate the enzyme activity by naked eyes. Our study not only expands the application of molecular self-assembly but also provides a useful method that can be applied for direct detection of enzyme activity in complex biological samples such as blood and cell lysates. PMID:27345959

  12. Two-dimensional magnetotransport in a black phosphorus naked quantum well

    PubMed Central

    Tayari, V.; Hemsworth, N.; Fakih, I.; Favron, A.; Gaufrès, E.; Gervais, G.; Martel, R.; Szkopek, T.

    2015-01-01

    Black phosphorus (bP) is the second known elemental allotrope with a layered crystal structure that can be mechanically exfoliated to atomic layer thickness. Unlike metallic graphite and semi-metallic graphene, bP is a semiconductor in both bulk and few-layer form. Here we fabricate bP-naked quantum wells in a back-gated field effect transistor geometry with bP thicknesses ranging from 6±1 nm to 47±1 nm. Using a polymer encapsulant, we suppress bP oxidation and observe field effect mobilities up to 900 cm2 V−1 s−1 and on/off current ratios exceeding 105. Shubnikov-de Haas oscillations observed in magnetic fields up to 35 T reveal a 2D hole gas with Schrödinger fermion character in a surface accumulation layer. Our work demonstrates that 2D electronic structure and 2D atomic structure are independent. 2D carrier confinement can be achieved without approaching atomic layer thickness, advantageous for materials that become increasingly reactive in the few-layer limit such as bP. PMID:26151889

  13. Two-dimensional magnetotransport in a black phosphorus naked quantum well.

    PubMed

    Tayari, V; Hemsworth, N; Fakih, I; Favron, A; Gaufrès, E; Gervais, G; Martel, R; Szkopek, T

    2015-01-01

    Black phosphorus (bP) is the second known elemental allotrope with a layered crystal structure that can be mechanically exfoliated to atomic layer thickness. Unlike metallic graphite and semi-metallic graphene, bP is a semiconductor in both bulk and few-layer form. Here we fabricate bP-naked quantum wells in a back-gated field effect transistor geometry with bP thicknesses ranging from 6±1 nm to 47±1 nm. Using a polymer encapsulant, we suppress bP oxidation and observe field effect mobilities up to 900 cm(2) V(-1) s(-1) and on/off current ratios exceeding 10(5). Shubnikov-de Haas oscillations observed in magnetic fields up to 35 T reveal a 2D hole gas with Schrödinger fermion character in a surface accumulation layer. Our work demonstrates that 2D electronic structure and 2D atomic structure are independent. 2D carrier confinement can be achieved without approaching atomic layer thickness, advantageous for materials that become increasingly reactive in the few-layer limit such as bP. PMID:26151889

  14. Naked-Eye Coulometric Sensor Using a Longitudinally Oriented Ag Band Electrode in a Microfluidic Channel.

    PubMed

    Oh, Jung-Min; Chow, Kwok-Fan

    2016-05-01

    In this Article, we report a coulometric sensing platform that is capable of sensing analytes on a working electrode and providing a visual readout of the analyte concentration on a silver (Ag) band counter electrode in a microchannel. The display mechanism relies on the electro-oxidation of metallic Ag as a complementary reaction to the sensing reduction reaction. The Ag band counter electrode is arranged longitudinally in a microchannel while the frontal tip of the band electrode directly faces a gold (Au) working electrode, which lies across the microchannel. The Ag oxidation always occurs at the band electrode's tip region that faces the working electrode due to the Ohmic potential drop across the solution in the microchannel. The decrement of the Ag electrode, which is clearly measurable with the naked eye, correlates linearly with an analyte concentration (e.g., 0.1-2.5 mM p-benzoquinone) and with an analyte feeding rate (i.e., a sample solution flow rate of 1.0-75.0 μL min(-1)). The platform design is also effective for a model analyte of horseradish peroxidase (HRP)-avidin in the dynamic range of 0.1-3.0 μg mL(-1). PMID:27064358

  15. Tumour resistance in induced pluripotent stem cells derived from naked mole-rats.

    PubMed

    Miyawaki, Shingo; Kawamura, Yoshimi; Oiwa, Yuki; Shimizu, Atsushi; Hachiya, Tsuyoshi; Bono, Hidemasa; Koya, Ikuko; Okada, Yohei; Kimura, Tokuhiro; Tsuchiya, Yoshihiro; Suzuki, Sadafumi; Onishi, Nobuyuki; Kuzumaki, Naoko; Matsuzaki, Yumi; Narita, Minoru; Ikeda, Eiji; Okanoya, Kazuo; Seino, Ken-Ichiro; Saya, Hideyuki; Okano, Hideyuki; Miura, Kyoko

    2016-01-01

    The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype-ARF suppression-induced senescence (ASIS)-that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR. PMID:27161380

  16. A Rapid In Situ Colorimetric Assay for Cobalt Detection by the Naked Eye.

    PubMed

    Kang, Sung-Min; Jang, Sung-Chan; Kim, Gi Yong; Lee, Chang-Soo; Huh, Yun Suk; Roh, Changhyun

    2016-01-01

    A simple, rapid, and convenient colorimetric chemosensor of a specific target toward the end user is still required for on-site detection and real-time monitoring applications. In this study, we developed a rapid in situ colorimetric assay for cobalt detection using the naked eye. Interestingly, a yellow to light orange visual color transition was observed within 3 s when a Chrysoidine G (CG) chemosensor was exposed to cobalt. Surprisingly, the CG chemosensor had great selectivity toward cobalt without any interference of other metal ions. Under optimized conditions, a lower detection limit of 0.1 ppm via a spectrophotometer and a visual detection limit of 2 ppm with a linear range from 0.4 to 1 ppm (R² = 0.97) were determined. Moreover, the CG chemosensor is reversible and maintains its functionality after treatment with chelating agents. In conclusion, we show the superior capabilities of the CG chemosensor, which has the potential to provide extremely facile handling, high sensitivity, and a fast response time for applications of on-site detection to real-time cobalt monitoring for the general public. PMID:27144568

  17. Hydrogen production by the naked active site of the di-iron hydrogenases in water.

    PubMed

    Zipoli, Federico; Car, Roberto; Cohen, Morrel H; Selloni, Annabella

    2009-10-01

    We explored the reactivity of the active center of the [FeFe]-hydrogenases detached from the enzyme and immersed in acidified water by first-principles Car-Parrinello molecular-dynamics simulations. We focused on the identification of the structures that are stable and metastable in acidified water and on their activity for hydrogen production. Our calculations revealed that the naked active center could be an efficient catalyst provided that electrons are transferred to the cluster. We found that both bridging and terminal isomers are present at equilibrium and that the bridging configuration is essential for efficient hydrogen production. The formation of the hydrogen molecule occurs via sequential protonations of the distal iron and of the N-atom of the S-CH(2)-NH-CH(2)-S chelating group. H(2) desorption does not involve a significant energy barrier, making the process very efficient at room temperature. We established that the bottleneck in the reaction is the direct proton transfer from water to the vacant site of the distal iron. Moreover, we found that even if the terminal isomer is present at the equilibrium, its strong local hydrophobicity prevents poisoning of the cluster. PMID:19737003

  18. Simultaneous Detection of Multiple Fish Pathogens Using a Naked-Eye Readable DNA Microarray

    PubMed Central

    Chang, Chin-I; Hung, Pei-Hsin; Wu, Chia-Che; Cheng, Ta Chih; Tsai, Jyh-Ming; Lin, King-Jung; Lin, Chung-Yen

    2012-01-01

    We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3′-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 103 CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens. PMID:22736973

  19. Ultra-low-energy (<10 eV/u) ion beam bombardment effect on naked DNA

    NASA Astrophysics Data System (ADS)

    Thopan, P.; Thongkumkoon, P.; Prakrajang, K.; Suwannakachorn, D.; Yu, L. D.

    2014-05-01

    Since ion energy deposition in the ion-bombarded materials dominantly occurs in the low-energy range, it is very interesting to know effects from ultra-low-energy ion interaction with DNA for understanding ion-beam-induced genetic mutation. Tens-keV Ar- and N-ion beams were decelerated to ultra-low energy ranging from 20 to 100 eV, or only a few to 10 eV/u, to bombard naked plasmid DNA. The bombarded DNA was analyzed using gel electrophoresis for DNA form changes. The original DNA supercoiled form was found to change to relaxed and linear forms, indicating single or double strand breaks after bombarded by tens-eV ion beam. N-ion beam was found more effective in inducing DNA change and mutation than Ar-ion beam. The study demonstrated that the ion bombardment with energy as low as several-tens eV was able to break DNA strands and thus potentially to cause genetic modification of biological cells. The experimental results were discussed in terms of direct atomic collision between the ions and DNA atoms.

  20. Tumour resistance in induced pluripotent stem cells derived from naked mole-rats

    PubMed Central

    Miyawaki, Shingo; Kawamura, Yoshimi; Oiwa, Yuki; Shimizu, Atsushi; Hachiya, Tsuyoshi; Bono, Hidemasa; Koya, Ikuko; Okada, Yohei; Kimura, Tokuhiro; Tsuchiya, Yoshihiro; Suzuki, Sadafumi; Onishi, Nobuyuki; Kuzumaki, Naoko; Matsuzaki, Yumi; Narita, Minoru; Ikeda, Eiji; Okanoya, Kazuo; Seino, Ken-ichiro; Saya, Hideyuki; Okano, Hideyuki; Miura, Kyoko

    2016-01-01

    The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype—ARF suppression-induced senescence (ASIS)—that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR. PMID:27161380

  1. Naked-eye cadmium sensor: using chromoionophore arrays of Langmuir-Blodgett molecular assemblies.

    PubMed

    Prabhakaran, Deivasigamani; Yuehong, Ma; Nanjo, Hiroshi; Matsunaga, Hideyuki

    2007-06-01

    This study demonstrates the possibility of a reversible naked-eye detection method for submicromolar levels of cadmium(II) using the Langmuir-Blodgett (L-B) technique. Molecular assemblies of 4-n-dodecyl-6-(2-thiazolylazo)resorcinol are transferred on precleaned microscopic glass slides, to act as a sensing probe. Isotherm (pi-A) measurements were performed to ensure the films' structural rigidity and homogeneity during sensor fabrication. The sensor surface morphology was characterized using atomic force microscopy and scanning electron microscopy. The probe membrane exhibits visual color transition, forming a series of reddish-orange to pinkish-purple complexes with cadmium, over a wide concentration range (0.04-44.5 microM). Cadmium response kinetics and the changes in the sensors' intrinsic optical properties were monitored using absorption spectroscopy and further confirmed using X-ray photoelectron spectroscopy. A hybrid L-B film composite of poly(vinyl stearate) and poly(vinyl-N-octadecylcarbamate) were investigated for enhancing sensor performance. The sensor was tested for its practical approach to prove its cadmium selectivity and sensitivity amid common matrix constituents using synthetic mixtures and real water samples. Using the sensor strips, the respective lower limits of cadmium detection and quantification are 0.039 and 0.050 microM, as estimated from a normalized linear calibration plot. PMID:17447727

  2. Carbon dots for naked eye colorimetric ultrasensitive arsenic and glutathione detection.

    PubMed

    Gupta, Abhishek; Verma, Navneet Chandra; Khan, Syamantak; Nandi, Chayan Kanti

    2016-07-15

    A novel one-step method for the synthesis of bright, multicolor fluorescent sulphur doped carbon dots (CNDs) has been developed by using simple microwave assisted pyrolysis of citric acid and sodium thiosulphate. The synthesized CNDs showed dual mode naked eye colorimetric ultrasensitive sensing capability both for arsenic [As (III)] and glutathione (GSH) with high selectivity. Using fluorometric assay, the detection limit (DL) for As (III) was found to be as low as 32pM. The selectivity data show that the newly developed CNDs is very specific for As (III) even with interference by high concentrations of other metal ions. The CNDs were also able to detect GSH very selectively over other biothiols like cysteine (Cys) and homo-cysteine (H-cys) with a DL of 43nM, even in blood plasma. The fast kinetic data suggests that the present CNDs assay could be used onsite As (III) detection. The CNDs, further, showed its potential application in high resolution bioimaging of bacterial nucleoid segregation. PMID:27015150

  3. A search for evidence of cold dust around naked T Tauri stars

    NASA Technical Reports Server (NTRS)

    Skinner, Stephen L.; Brown, Alexander; Walter, Frederick M.

    1991-01-01

    Results of millimeter/ submillimeter photometry of 16 naked T Tauri stars, 11 of which lie in the nearby Taurus-Auriga star-forming region, are presented. Evidence of cold circumstellar dust, which could exist around these objects in the form of remnant disks if NTTS evolved from classical T Tauri stars, is explored. The only star detected at the primary observing wavelength of 1100 microns was V836 Tau, which was also detected at 800 microns with an upper limit obtained at 450 microns. Its spectral energy distribution longward of 10 microns is consistent with that expected for a flat axisymmetric moderately active disk with a solar mass of 0.04. Upper limits on the disk masses of undetected stars in Taurus-Auriga are typically 0.02 solar mass, for an assumed temperature of not less than 6 K at the outer disk radius. It is argued that the high levels of magnetic activity found in NTTS may produce detectable gyrosynchroton emission at millimeter wavelengths.

  4. The traumatic element in the typical dream of feeling embarrassed at being naked.

    PubMed

    Myers, W A

    1989-01-01

    Material is presented from three cases, where analysis of repetitive dreams of feeling embarrassment at being partially or totally naked was an important feature of the treatment. The indifference by the other people in the dream to the dreamer's nakedness was initially linked to perceived transference slights at the hands of the analyst, and later to repeated episodes of actually being treated indifferently at the hands of the parents. This indifference was related to latency or adolescent attempts by the patients to gain love or attention from the parents by exhibitionistic means. The stereotypical presentation of the manifest content of these dreams is seen as evidence for their underlying traumatic roots. Such dreams are likened to the typical examination dreams described by Freud, which have also been noted by others to have traumatic roots. This finding is consistent with my own work with certain repetitive manifest dream configurations and with Freud's (1920) reevaluation of his theory of dreams in Beyond the Pleasure Principle, wherein he noted that dreams of patients suffering from traumatic neurosis often manifestly repeated the traumatic situation in an attempt to master it retrospectively. PMID:2708770

  5. A Rapid In Situ Colorimetric Assay for Cobalt Detection by the Naked Eye

    PubMed Central

    Kang, Sung-Min; Jang, Sung-Chan; Kim, Gi Yong; Lee, Chang-Soo; Huh, Yun Suk; Roh, Changhyun

    2016-01-01

    A simple, rapid, and convenient colorimetric chemosensor of a specific target toward the end user is still required for on-site detection and real-time monitoring applications. In this study, we developed a rapid in situ colorimetric assay for cobalt detection using the naked eye. Interestingly, a yellow to light orange visual color transition was observed within 3 s when a Chrysoidine G (CG) chemosensor was exposed to cobalt. Surprisingly, the CG chemosensor had great selectivity toward cobalt without any interference of other metal ions. Under optimized conditions, a lower detection limit of 0.1 ppm via a spectrophotometer and a visual detection limit of 2 ppm with a linear range from 0.4 to 1 ppm (R2 = 0.97) were determined. Moreover, the CG chemosensor is reversible and maintains its functionality after treatment with chelating agents. In conclusion, we show the superior capabilities of the CG chemosensor, which has the potential to provide extremely facile handling, high sensitivity, and a fast response time for applications of on-site detection to real-time cobalt monitoring for the general public. PMID:27144568

  6. Cerebellar Expression of the Neurotrophin Receptor p75 in Naked-Ataxia Mutant Mouse

    PubMed Central

    Rahimi Balaei, Maryam; Jiao, Xiaodan; Ashtari, Niloufar; Afsharinezhad, Pegah; Ghavami, Saeid; Marzban, Hassan

    2016-01-01

    Spontaneous mutation in the lysosomal acid phosphatase 2 (Acp2) mouse (nax—naked-ataxia mutant mouse) correlates with severe cerebellar defects including ataxia, reduced size and abnormal lobulation as well as Purkinje cell (Pc) degeneration. Loss of Pcs in the nax cerebellum is compartmentalized and harmonized to the classic pattern of gene expression of the cerebellum in the wild type mouse. Usually, degeneration starts in the anterior and posterior zones and continues to the central and nodular zones of cerebellum. Studies have suggested that the p75 neurotrophin receptor (NTR) plays a role in Pc degeneration; thus, in this study, we investigated the p75NTR pattern and protein expression in the cerebellum of the nax mutant mouse. Despite massive Pc degeneration that was observed in the nax mouse cerebellum, p75NTR pattern expression was similar to the HSP25 pattern in nax mice and comparable with wild type sibling cerebellum. In addition, immunoblot analysis of p75NTR protein expression did not show any significant difference between nax and wild type sibling (p > 0.5). In comparison with wild type counterparts, p75NTR pattern expression is aligned with the fundamental cytoarchitecture organization of the cerebellum and is unchanged in the nax mouse cerebellum despite the severe neurodevelopmental disorder accompanied with Pc degeneration. PMID:26784182

  7. A Novel Thermal Electrochemical Synthesis Method for Production of Stable Colloids of "Naked" Metal (Ag) Nanocrystals

    SciTech Connect

    Hu, Michael Z.; Easterly, Clay E

    2009-01-01

    Solution synthesis of nanocrystal silver is reviewed. This paper reports a novel thermal electrochemical synthesis (TECS) for producing metal Ag nanocrystals as small as a few nanometers. The TECS method requires mild conditions (25-100oC), low voltage (1-50 V DC) on Ag electrodes, and simple water or aqueous solutions as reaction medium. Furthermore, a tubular dialysis membrane surround electrodes proves favorable to produce nanosized (<10 nm) Ag nanocrystals. Different from those nanocrystals reported in literature, our nanocrystals have several unique features: (1) small nanometer size, (2) nakedness , i.e., surfaces of metal nanocrystals are free of organic ligands or capping molecules and no need of dispersant in synthesis solutions, and (3) colloidally stable in water solutions. It was discovered that Ag nanoparticles with initially large size distribution can be homogenized into near-monodispersed system by a low power (< 15 mW) He-Ne laser exposure treatment. The combination of the TECS technique and the laser treatment could lead to a new technology that produces metal nanoparticles that are naked, stable, and uniform sized. In the presence of stabilizing agent (also as supporting electrolyte) such as polyvinyl alcohol (PVA), large yield of silver nanoparticles (<100nm) in the form of thick milky sols are produced.

  8. Simultaneous detection of multiple fish pathogens using a naked-eye readable DNA microarray.

    PubMed

    Chang, Chin-I; Hung, Pei-Hsin; Wu, Chia-Che; Cheng, Ta Chih; Tsai, Jyh-Ming; Lin, King-Jung; Lin, Chung-Yen

    2012-01-01

    We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 10(3) CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens. PMID:22736973

  9. Challenging the inbreeding hypothesis in a eusocial mammal: population genetics of the naked mole-rat, Heterocephalus glaber.

    PubMed

    Ingram, Colleen M; Troendle, Nicholas J; Gill, Clare A; Braude, Stanton; Honeycutt, Rodney L

    2015-10-01

    The role of genetic relatedness in the evolution of eusociality has been the topic of much debate, especially when contrasting eusocial insects with vertebrates displaying reproductive altruism. The naked mole-rat, Heterocephalus glaber, was the first described eusocial mammal. Although this discovery was based on an ecological constraints model of eusocial evolution, early genetic studies reported high levels of relatedness in naked mole-rats, providing a compelling argument that low dispersal rates and consanguineous mating (inbreeding as a mating system) are the driving forces for the evolution of this eusocial species. One caveat to accepting this long-held view is that the original genetic studies were based on limited sampling from the species' geographic distribution. A growing body of evidence supports a contrary view, with the original samples not representative of the species-rather reflecting a single founder event, establishing a small population south of the Athi River. Our study is the first to address these competing hypotheses by examining patterns of molecular variation in colonies sampled from north and south of the Athi and Tana rivers, which based on our results, serve to isolate genetically distinct populations of naked mole-rats. Although colonies south of the Athi River share a single mtDNA haplotype and are fixed at most microsatellite loci, populations north of the Athi River are considerably more variable. Our findings support the position that the low variation observed in naked mole-rat populations south of the Athi River reflects a founder event, rather than a consequence of this species' unusual mating system. PMID:26407630

  10. Cosmic censorship and stationary states of half-spin particles in the field of Reissner-Nordström naked singularity

    NASA Astrophysics Data System (ADS)

    Gorbatenko, M. V.; Neznamov, V. P.; Popov, E. Yu; Safronov, I. I.

    2016-02-01

    The paper explores quantum mechanics of half-spin particle motion in the field of Reissner-Nordström (RN) naked singularity. It is shown that for any quantum mechanical Dirac particle, irrespective of availability and sign of its electrical charge, the RN naked singularity is separated by an infinitely high positive potential barrier. With like charges of a particle and the source of the RN naked singularity, near the origin there exists the second completely impenetrable potential barrier. It has been proved that in the field of the RN naked singularity, bound states of half-spin particles can exist. The conditions for appearance of such states were revealed and computations were performed to find energy eigenvalues and eigenfunctions.

  11. Kinetics of poliovirus shedding following oral vaccination as measured by quantitative reverse transcription-PCR versus culture.

    PubMed

    Taniuchi, Mami; Begum, Sharmin; Uddin, Md Jashim; Platts-Mills, James A; Liu, Jie; Kirkpatrick, Beth D; Chowdhury, Anwarul H; Jamil, Khondoker M; Haque, Rashidul; Petri, William A; Houpt, Eric R

    2015-01-01

    Amid polio eradication efforts, detection of oral polio vaccine (OPV) virus in stool samples can provide information about rates of mucosal immunity and allow estimation of the poliovirus reservoir. We developed a multiplex one-step quantitative reverse transcription-PCR (qRT-PCR) assay for detection of OPV Sabin strains 1, 2, and 3 directly in stool samples with an external control to normalize samples for viral quantity and compared its performance with that of viral culture. We applied the assay to samples from infants in Dhaka, Bangladesh, after the administration of trivalent OPV (tOPV) at weeks 14 and 52 of life (on days 0 [pre-OPV], +4, +11, +18, and +25 relative to vaccination). When 1,350 stool samples were tested, the sensitivity and specificity of the quantitative PCR (qPCR) assay were 89 and 91% compared with culture. A quantitative relationship between culture(+)/qPCR(+) and culture(-)/qPCR(+) stool samples was observed. The kinetics of shedding revealed by qPCR and culture were similar. qPCR quantitative cutoffs based on the day +11 or +18 stool samples could be used to identify the culture-positive shedders, as well as the long-duration or high-frequency shedders. Interestingly, qPCR revealed that a small minority (7%) of infants contributed the vast majority (93 to 100%) of the total estimated viral excretion across all subtypes at each time point. This qPCR assay for OPV can simply and quantitatively detect all three Sabin strains directly in stool samples to approximate shedding both qualitatively and quantitatively. PMID:25378579

  12. Poliovirus Internal Ribosome Entry Segment Structure Alterations That Specifically Affect Function in Neuronal Cells: Molecular Genetic Analysis

    PubMed Central

    Malnou, Cécile E.; Pöyry, Tuija A. A.; Jackson, Richard J.; Kean, Katherine M.

    2002-01-01

    Translation of poliovirus RNA is driven by an internal ribosome entry segment (IRES) present in the 5′ noncoding region of the genomic RNA. This IRES is structured into several domains, including domain V, which contains a large lateral bulge-loop whose predicted secondary structure is unclear. The primary sequence of this bulge-loop is strongly conserved within enteroviruses and rhinoviruses: it encompasses two GNAA motifs which could participate in intrabulge base pairing or (in one case) could be presented as a GNRA tetraloop. We have begun to address the question of the significance of the sequence conservation observed among enterovirus reference strains and field isolates by using a comprehensive site-directed mutagenesis program targeted to these two GNAA motifs. Mutants were analyzed functionally in terms of (i) viability and growth kinetics in both HeLa and neuronal cell lines, (ii) structural analyses by biochemical probing of the RNA, and (iii) translation initiation efficiencies in vitro in rabbit reticulocyte lysates supplemented with HeLa or neuronal cell extracts. Phenotypic analyses showed that only viruses with both GNAA motifs destroyed were significantly affected in their growth capacities, which correlated with in vitro translation defects. The phenotypic defects were strongly exacerbated in neuronal cells, where a temperature-sensitive phenotype could be revealed at between 37 and 39.5°C. Biochemical probing of mutated domain V, compared to the wild type, demonstrated that such mutations lead to significant structural perturbations. Interestingly, revertant viruses possessed compensatory mutations which were distant from the primary mutations in terms of sequence and secondary structure, suggesting that intradomain tertiary interactions could exist within domain V of the IRES. PMID:12368304

  13. Circular geodesics of naked singularities in the Kehagias-Sfetsos metric of Hořava's gravity

    NASA Astrophysics Data System (ADS)

    Vieira, Ronaldo S. S.; Schee, Jan; Kluźniak, Włodek; Stuchlík, Zdeněk; Abramowicz, Marek

    2014-07-01

    We discuss photon and test-particle orbits in the Kehagias-Sfetsos (KS) metric of Hořava's gravity. For any value of the Hořava parameter ω, there are values of the gravitational mass M for which the metric describes a naked singularity, and this is always accompanied by a vacuum "antigravity sphere" on whose surface a test particle can remain at rest (in a zero angular momentum geodesic), and inside which no circular geodesics exist. The observational appearance of an accreting KS naked singularity in a binary system would be that of a quasistatic spherical fluid shell surrounded by an accretion disk, whose properties depend on the value of M, but are always very different from accretion disks familiar from the Kerr-metric solutions. The properties of the corresponding circular orbits are qualitatively similar to those of the Reissner-Nordström naked singularities. When event horizons are present, the orbits outside the Kehagias-Sfetsos black hole are qualitatively similar to those of the Schwarzschild metric.

  14. Physical properties of naked DNA influence nucleosome positioning and correlate with transcription start and termination sites in yeast

    PubMed Central

    2011-01-01

    Background In eukaryotic organisms, DNA is packaged into chromatin structure, where most of DNA is wrapped into nucleosomes. DNA compaction and nucleosome positioning have clear functional implications, since they modulate the accessibility of genomic regions to regulatory proteins. Despite the intensive research effort focused in this area, the rules defining nucleosome positioning and the location of DNA regulatory regions still remain elusive. Results Naked (histone-free) and nucleosomal DNA from yeast were digested by microccocal nuclease (MNase) and sequenced genome-wide. MNase cutting preferences were determined for both naked and nucleosomal DNAs. Integration of their sequencing profiles with DNA conformational descriptors derived from atomistic molecular dynamic simulations enabled us to extract the physical properties of DNA on a genomic scale and to correlate them with chromatin structure and gene regulation. The local structure of DNA around regulatory regions was found to be unusually flexible and to display a unique pattern of nucleosome positioning. Ab initio physical descriptors derived from molecular dynamics were used to develop a computational method that accurately predicts nucleosome enriched and depleted regions. Conclusions Our experimental and computational analyses jointly demonstrate a clear correlation between sequence-dependent physical properties of naked DNA and regulatory signals in the chromatin structure. These results demonstrate that nucleosome positioning around TSS (Transcription Start Site) and TTS (Transcription Termination Site) (at least in yeast) is strongly dependent on DNA physical properties, which can define a basal regulatory mechanism of gene expression. PMID:21981773

  15. Discovery of naked charm particles and lifetime differences among charm species using nuclear emulsion techniques innovated in Japan

    PubMed Central

    NIU, Kiyoshi

    2008-01-01

    This is a historical review of the discovery of naked charm particles and lifetime differences among charm species. These discoveries in the field of cosmic-ray physics were made by the innovation of nuclear emulsion techniques in Japan. A pair of naked charm particles was discovered in 1971 in a cosmic-ray interaction, three years prior to the discovery of the hidden charm particle, J/Ψ, in western countries. Lifetime differences between charged and neutral charm particles were pointed out in 1975, which were later re-confirmed by the collaborative Experiment E531 at Fermilab. Japanese physicists led by K.Niu made essential contributions to it with improved emulsion techniques, complemented by electronic detectors. This review also discusses the discovery of artificially produced naked charm particles by us in an accelerator experiment at Fermilab in 1975 and of multiple-pair productions of charm particles in a single interaction in 1987 by the collaborative Experiment WA75 at CERN. PMID:18941283

  16. Flat band potential measurements of naked and viologen-modified n-WS[sub 2] electrodes in aqueous iodide and triiodide solutions

    SciTech Connect

    Huang, J.; Wrighton, M.S. )

    1994-09-15

    The flat band potentials, E[sub FB], of naked n-WS[sub 2] electrodes and cationic viologen polymer-modified n-WS[sub 2] electrodes have been determined in KI and KI[sub 3] solutions by differential capacitance measurements. The E[sub FB] values for naked n-WS[sub 2] electrodes are shifted negatively in electrolyte media containing I[sup [minus

  17. GRB 080503: IMPLICATIONS OF A NAKED SHORT GAMMA-RAY BURST DOMINATED BY EXTENDED EMISSION

    SciTech Connect

    Perley, D. A.; Metzger, B. D.; Butler, N. R.; Bloom, J. S.; Miller, A. A.; Filippenko, A. V.; Li, W.; Granot, J.; Sakamoto, T.; Gehrels, N.; Ramirez-Ruiz, E.; Bunker, A.; Chen, H.-W.; Glazebrook, K.; Hall, P. B.; Hurley, K. C.; Kocevski, D.; Norris, J.

    2009-05-10

    We report on observations of GRB 080503, a short gamma-ray burst (GRB) with very bright extended emission (about 30 times the gamma-ray fluence of the initial spike) in conjunction with a thorough comparison to other short Swift events. In spite of the prompt-emission brightness, however, the optical counterpart is extraordinarily faint, never exceeding 25 mag in deep observations starting at {approx}1 hr after the Burst Alert Telescope (BAT) trigger. The optical brightness peaks at {approx}1 day and then falls sharply in a manner similar to the predictions of Li and Paczynski (1998) for supernova-like emission following compact binary mergers. However, a shallow spectral index and similar evolution in X-rays inferred from Chandra observations are more consistent with an afterglow interpretation. The extreme faintness of this probable afterglow relative to the bright gamma-ray emission argues for a very low density medium surrounding the burst (a 'naked' GRB), consistent with the lack of a coincident host galaxy down to 28.5 mag in deep Hubble Space Telescope imaging. The late optical and X-ray peak could be explained by a slightly off-axis jet or by a refreshed shock. Our observations reinforce the notion that short GRBs generally occur outside regions of active star formation, but demonstrate that in some cases the luminosity of the extended prompt emission can greatly exceed that of the short spike, which may constrain theoretical interpretation of this class of events. This extended emission is not the onset of an afterglow, and its relative brightness is probably either a viewing-angle effect or intrinsic to the central engine itself. Because most previous BAT short bursts without observed extended emission are too faint for this signature to have been detectable even if it were present at typical level, conclusions based solely on the observed presence or absence of extended emission in the existing Swift sample are premature.

  18. CONSTRAINTS ON THE EMISSION MODEL OF THE 'NAKED-EYE BURST' GRB 080319B

    SciTech Connect

    Abdo, A. A.; Abeysekara, A. U.; Linnemann, J. T.; Allen, B. T.; Chen, C.; Aune, T.; Berley, D.; Goodman, J. A.; Christopher, G. E.; Kolterman, B. E.; Mincer, A. I.; DeYoung, T.; Dingus, B. L.; Hoffman, C. M.; Ellsworth, R. W.; Gonzalez, M. M.; Granot, J.; Hays, E.; McEnery, J. E.; Huentemeyer, P. H.; and others

    2012-07-10

    On 2008 March 19, one of the brightest gamma-ray bursts (GRBs) ever recorded was detected by several ground- and space-based instruments spanning the electromagnetic spectrum from radio to gamma rays. With a peak visual magnitude of 5.3, GRB 080319B was dubbed the 'naked-eye' GRB, as an observer under dark skies could have seen the burst without the aid of an instrument. Presented here are results from observations of the prompt phase of GRB 080319B taken with the Milagro TeV observatory. The burst was observed at an elevation angle of 47 Degree-Sign . Analysis of the data is performed using both the standard air shower method and the scaler or single-particle technique, which results in a sensitive energy range that extends from {approx}5 GeV to >20 TeV. These observations provide the only direct constraints on the properties of the high-energy gamma-ray emission from GRB 080319B at these energies. No evidence for emission is found in the Milagro data, and upper limits on the gamma-ray flux above 10 GeV are derived. The limits on emission between {approx}25 and 200 GeV are incompatible with the synchrotron self-Compton model of gamma-ray production and disfavor a corresponding range (2 eV-16 eV) of assumed synchrotron peak energies. This indicates that the optical photons and soft ({approx}650 keV) gamma rays may not be produced by the same electron population.

  19. Q2122-444: A NAKED ACTIVE GALACTIC NUCLEUS FULLY DRESSED

    SciTech Connect

    Gliozzi, M.; Satyapal, S.; Panessa, F.; Franca, F. La; Saviane, I.; Monaco, L.; Foschini, L.; Kedziora-Chudczer, L.; Sambruna, R. M.

    2010-12-20

    Based on previous spectral and temporal optical studies, Q2122-444 has been classified as a naked active galactic nucleus (AGN) or true type 2 AGN, that is, an AGN that genuinely lacks a broad-line region (BLR). Its optical spectrum seemed to possess only narrow forbidden emission lines that are typical of type 2 (obscured) AGNs, but the long-term optical light curve, obtained from a monitoring campaign over more than two decades, showed strong variability, apparently ruling out the presence of heavy obscuration. Here we present the results from a {approx}40 ks XMM-Newton observation of Q2122-444 carried out to shed light on the energetics of this enigmatic AGN. The X-ray analysis was complemented with Australia Telescope Compact Array radio data to assess the possible presence of a jet, and with new NTT/EFOSC2 optical spectroscopic data to verify the actual absence of a BLR. The higher-quality optical data revealed the presence of strong and broad Balmer lines that are at odds with the previous spectral classification of this AGN. The lack of detection of radio emission rules out the presence of a jet. The X-ray data combined with simultaneous UV observations carried out by the Optical Monitor (OM) aboard XMM-Newton confirm that Q2122-444 is a typical type 1 AGN without any significant intrinsic absorption. New estimates of the black hole mass independently obtained from the broad Balmer lines and from a new scaling technique based on X-ray spectral data suggest that Q2122-444 is accreting at a relatively high rate in Eddington units.

  20. THE NAKED EMERGENCE OF SOLAR ACTIVE REGIONS OBSERVED WITH SDO/HMI

    SciTech Connect

    Centeno, Rebecca

    2012-11-01

    We take advantage of the HMI/SDO instrument to study the naked emergence of active regions (ARs) from the first imprints of the magnetic field on the solar surface. To this end, we followed the first 24 hr in the life of two rather isolated ARs that appeared on the surface when they were about to cross the central meridian. We analyze the correlations between Doppler velocities and the orientation of the vector magnetic field, consistent finding that the horizontal fields connecting the main polarities are dragged to the surface by relatively strong upflows and are associated with elongated granulation that is, on average, brighter than its surroundings. The main magnetic footpoints, on the other hand, are dominated by vertical fields and downflowing plasma. The appearance of moving dipolar features (MDFs, of opposite polarity to that of the AR) in between the main footpoints is a rather common occurrence once the AR reaches a certain size. The buoyancy of the fields is insufficient to lift up the magnetic arcade as a whole. Instead, weighted by the plasma that it carries, the field is pinned down to the photosphere at several places in between the main footpoints, giving life to the MDFs and enabling channels of downflowing plasma. MDF poles tend to drift toward each other, merge and disappear. This is likely to be the signature of a reconnection process in the dipped field lines, which relieves some of the weight allowing the magnetic arcade to finally rise beyond the detection layer of the Helioseismic and Magnetic Imager spectral line.

  1. The Insulin-Like Growth Factor System in the Long-Lived Naked Mole-Rat

    PubMed Central

    Brohus, Malene; Gorbunova, Vera; Faulkes, Chris G.; Overgaard, Michael T.; Conover, Cheryl A.

    2015-01-01

    Naked mole-rats (Heterocephalus glaber) (NMRs) are the longest living rodents known. They show negligible senescence, and are resistant to cancers and certain damaging effects associated with aging. The insulin-like growth factors (IGFs) have pluripotent actions, influencing growth processes in virtually every system of the body. They are established contributors to the aging process, confirmed by the demonstration that decreased IGF signaling results in life-extending effects in a variety of species. The IGFs are likewise involved in progression of cancers by mediating survival signals in malignant cells. This report presents a full characterization of the IGF system in the NMR: ligands, receptors, IGF binding proteins (IGFBPs), and IGFBP proteases. A particular emphasis was placed on the IGFBP protease, pregnancy-associated plasma protein-A (PAPP-A), shown to be an important lifespan modulator in mice. Comparisons of IGF-related genes in the NMR with human and murine sequences indicated no major differences in essential parts of the IGF system, including PAPP-A. The protease was shown to possess an intact active site despite the report of a contradictory genome sequence. Furthermore, PAPP-A was expressed and translated in NMRs cells and retained IGF-dependent proteolytic activity towards IGFBP-4 and IGF-independent activity towards IGFBP-5. However, experimental data suggest differential regulatory mechanisms for PAPP-A expression in NMRs than those described in humans and mice. This overall description of the IGF system in the NMR represents an initial step towards elucidating the complex molecular mechanisms underlying longevity, and how these animals have evolved to ensure a delayed and healthy aging process. PMID:26694858

  2. The Insulin-Like Growth Factor System in the Long-Lived Naked Mole-Rat.

    PubMed

    Brohus, Malene; Gorbunova, Vera; Faulkes, Chris G; Overgaard, Michael T; Conover, Cheryl A

    2015-01-01

    Naked mole-rats (Heterocephalus glaber) (NMRs) are the longest living rodents known. They show negligible senescence, and are resistant to cancers and certain damaging effects associated with aging. The insulin-like growth factors (IGFs) have pluripotent actions, influencing growth processes in virtually every system of the body. They are established contributors to the aging process, confirmed by the demonstration that decreased IGF signaling results in life-extending effects in a variety of species. The IGFs are likewise involved in progression of cancers by mediating survival signals in malignant cells. This report presents a full characterization of the IGF system in the NMR: ligands, receptors, IGF binding proteins (IGFBPs), and IGFBP proteases. A particular emphasis was placed on the IGFBP protease, pregnancy-associated plasma protein-A (PAPP-A), shown to be an important lifespan modulator in mice. Comparisons of IGF-related genes in the NMR with human and murine sequences indicated no major differences in essential parts of the IGF system, including PAPP-A. The protease was shown to possess an intact active site despite the report of a contradictory genome sequence. Furthermore, PAPP-A was expressed and translated in NMRs cells and retained IGF-dependent proteolytic activity towards IGFBP-4 and IGF-independent activity towards IGFBP-5. However, experimental data suggest differential regulatory mechanisms for PAPP-A expression in NMRs than those described in humans and mice. This overall description of the IGF system in the NMR represents an initial step towards elucidating the complex molecular mechanisms underlying longevity, and how these animals have evolved to ensure a delayed and healthy aging process. PMID:26694858

  3. An ion beam deceleration lens for ultra-low-energy ion bombardment of naked DNA

    NASA Astrophysics Data System (ADS)

    Thopan, P.; Prakrajang, K.; Thongkumkoon, P.; Suwannakachorn, D.; Yu, L. D.

    2013-07-01

    Study of low-energy ion bombardment effect on biological living materials is of significance. High-energy ion beam irradiation of biological materials such as organs and cells has no doubt biological effects. However, ion energy deposition in the ion-bombarded materials dominantly occurs in the low-energy range. To investigate effects from very-low-energy ion bombardment on biological materials, an ion beam deceleration lens is necessary for uniform ion energy lower than keV. A deceleration lens was designed and constructed based on study of the beam optics using the SIMION program. The lens consisted of six electrodes, able to focus and decelerate primary ion beam, with the last one being a long tube to obtain a parallel uniform exiting beam. The deceleration lens was installed to our 30-kV bioengineering-specialized ion beam line. The final decelerated-ion energy was measured using a simple electrostatic field to bend the beam to range from 10 eV to 1 keV controlled by the lens parameters and the primary beam condition. In a preliminary test, nitrogen ion beam at 60 eV decelerated from a primary 20-keV beam bombarded naked plasmid DNA. The original DNA supercoiled form was found to change to relaxed and linear forms, indicating single or double strand breaks. The study demonstrated that the ion bombardment with energy as low as several-tens eV was possible to break DNA strands and thus potential to cause genetic modification of biological cells.

  4. Naked mole-rats: behavioural phenotyping and comparison with C57BL/6 mice.

    PubMed

    Deacon, R M J; Dulu, T D; Patel, N B

    2012-05-16

    Naked mole-rats (NMR) live underground in large eusocial colonies in East Africa. They are extremely long-lived, some individuals having a lifespan of over 30 years. This has attracted research into longevity and possibly neurodegenerative disorders. However, very little is known about their basic behaviour, particularly in tests commonly used to characterise the behaviour of the laboratory rat and mouse, for which there is an enormous database. Recently the authors carried out comprehensive behavioural phenotyping on NMRs, comparing them on most tasks directly with C57BL/6 mice, the strain for which there is the largest behavioural database. The NMR colony had been obtained from the wild originally, but housed in an animal facility for about two years. Large inter-species differences in behaviour were seen between the mice and the NMRs. The latter had generally poor sensorimotor function, including cutaneous sensation, strength and even grasp reflexes. They were often reluctant to enter or head-dip into small holes that mice readily entered. Their vision (generally considered to be very poor) was sufficient to distinguish the two zones of a light-dark box. Although, as expected, the NMRs were capable of burrowing and digging, when individually housed they did not shred cotton material to make nests. Shredding was seen in a colony cage containing a queen, but no nests were made there even when a nesting box was provided. In cognitive testing, although, unlike mice and rats, they did not spontaneously alternate in a T-maze, they learnt rewarded alternation and a cued position task well. This study demonstrates how behaviour uniquely reflects the natural environment in which these unusual animals have evolved and live, and provides baseline data for future work. PMID:22440234

  5. Initial Case Reports of Cancer in Naked Mole-rats (Heterocephalus glaber).

    PubMed

    Delaney, M A; Ward, J M; Walsh, T F; Chinnadurai, S K; Kerns, K; Kinsel, M J; Treuting, P M

    2016-05-01

    Naked mole-rats (NMRs;Heterocephalus glaber) are highly adapted, eusocial rodents renowned for their extreme longevity and resistance to cancer. Because cancer has not been formally described in this species, NMRs have been increasingly utilized as an animal model in aging and cancer research. We previously reported the occurrence of several age-related diseases, including putative pre-neoplastic lesions, in zoo-housed NMR colonies. Here, we report for the first time 2 cases of cancer in zoo-housed NMRs. In Case No. 1, we observed a subcutaneous mass in the axillary region of a 22-year-old male NMR, with histologic, immunohistochemical (pancytokeratin positive, rare p63 immunolabeling, and smooth muscle actin negative), and ultrastructural characteristics of an adenocarcinoma possibly of mammary or salivary origin. In Case No. 2, we observed a densely cellular, poorly demarcated gastric mass of polygonal cells arranged in nests with positive immunolabeling for synaptophysin and chromogranin indicative of a neuroendocrine carcinoma in an approximately 20-year-old male NMR. We also include a brief discussion of other proliferative growths and pre-cancerous lesions diagnosed in 1 zoo colony. Although these case reports do not alter the longstanding observation of cancer resistance, they do raise questions about the scope of cancer resistance and the interpretation of biomedical studies in this model. These reports also highlight the benefit of long-term disease investigations in zoo-housed populations to better understand naturally occurring disease processes in species used as models in biomedical research. PMID:26846576

  6. The Naked Emergence of Solar Active Regions Observed with SDO/HMI

    NASA Astrophysics Data System (ADS)

    Centeno, Rebecca

    2012-11-01

    We take advantage of the HMI/SDO instrument to study the naked emergence of active regions (ARs) from the first imprints of the magnetic field on the solar surface. To this end, we followed the first 24 hr in the life of two rather isolated ARs that appeared on the surface when they were about to cross the central meridian. We analyze the correlations between Doppler velocities and the orientation of the vector magnetic field, consistent finding that the horizontal fields connecting the main polarities are dragged to the surface by relatively strong upflows and are associated with elongated granulation that is, on average, brighter than its surroundings. The main magnetic footpoints, on the other hand, are dominated by vertical fields and downflowing plasma. The appearance of moving dipolar features (MDFs, of opposite polarity to that of the AR) in between the main footpoints is a rather common occurrence once the AR reaches a certain size. The buoyancy of the fields is insufficient to lift up the magnetic arcade as a whole. Instead, weighted by the plasma that it carries, the field is pinned down to the photosphere at several places in between the main footpoints, giving life to the MDFs and enabling channels of downflowing plasma. MDF poles tend to drift toward each other, merge and disappear. This is likely to be the signature of a reconnection process in the dipped field lines, which relieves some of the weight allowing the magnetic arcade to finally rise beyond the detection layer of the Helioseismic and Magnetic Imager spectral line.

  7. Transgenic mice as an alternative to monkeys for neurovirulence testing of live oral poliovirus vaccine: validation by a WHO collaborative study.

    PubMed Central

    Dragunsky, Eugenia; Nomura, Tatsuji; Karpinski, Kazimir; Furesz, John; Wood, David J.; Pervikov, Yuri; Abe, Shinobu; Kurata, Takeshi; Vanloocke, Olivier; Karganova, Galina; Taffs, Rolf; Heath, Alan; Ivshina, Anna; Levenbook, Inessa

    2003-01-01

    OBJECTIVE: Extensive WHO collaborative studies were performed to evaluate the suitability of transgenic mice susceptible to poliovirus (TgPVR mice, strain 21, bred and provided by the Central Institute for Experimental Animals, Japan) as an alternative to monkeys in the neurovirulence test (NVT) of oral poliovirus vaccine (OPV). METHODS: Nine laboratories participated in the collaborative study on testing neurovirulence of 94 preparations of OPV and vaccine derivatives of all three serotypes in TgPVR21 mice. FINDINGS: Statistical analysis of the data demonstrated that the TgPVR21 mouse NVT was of comparable sensitivity and reproducibility to the conventional WHO NVT in simians. A statistical model for acceptance/rejection of OPV lots in the mouse test was developed, validated, and shown to be suitable for all three vaccine types. The assessment of the transgenic mouse NVT is based on clinical evaluation of paralysed mice. Unlike the monkey NVT, histological examination of central nervous system tissue of each mouse offered no advantage over careful and detailed clinical observation. CONCLUSIONS: Based on data from the collaborative studies the WHO Expert Committee for Biological Standardization approved the mouse NVT as an alternative to the monkey test for all three OPV types and defined a standard implementation process for laboratories that wish to use the test. This represents the first successful introduction of transgenic animals into control of biologicals. PMID:12764491

  8. Rare natural type 3/type 2 intertypic capsid recombinant vaccine-related poliovirus isolated from a case of acute flaccid paralysis in Brazil, 2015.

    PubMed

    Cassemiro, Klécia M S M; Burlandy, Fernanda M; da Silva, Edson E

    2016-07-01

    A natural type 3/type 2 intertypic capsid recombinant vaccine-related poliovirus was isolated from an acute flaccid paralytic case in Brazil. Genome sequencing revealed the uncommon location of the crossover site in the VP1 coding region (nucleotides 3251-3258 of Sabin 3 genome). The Sabin 2 donor sequence replaced the last 118 nt of VP1, resulting in the substitution of the complete antigenic site IIIa by PV2-specific amino acids. The low overall number of nucleotide substitutions in P1 region indicated that the predicted replication time of the isolate was about 8-9 weeks. Two of the principal determinants of attenuation in Sabin 3 genomes were mutated (U472C and C2493U), but the temperature-sensitive phenotype of the isolate was preserved. Our results support the theory that there exists a PV3/PV2 recombination hotspot site in the tail region of the VP1 capsid protein and that the recombination may occur soon after oral poliovirus vaccine administration. PMID:27082658

  9. Conformational Ensemble of the Poliovirus 3CD Precursor Observed by MD Simulations and Confirmed by SAXS: A Strategy to Expand the Viral Proteome?

    PubMed Central

    Moustafa, Ibrahim M.; Gohara, David W.; Uchida, Akira; Yennawar, Neela; Cameron, Craig E.

    2015-01-01

    The genomes of RNA viruses are relatively small. To overcome the small-size limitation, RNA viruses assign distinct functions to the processed viral proteins and their precursors. This is exemplified by poliovirus 3CD protein. 3C protein is a protease and RNA-binding protein. 3D protein is an RNA-dependent RNA polymerase (RdRp). 3CD exhibits unique protease and RNA-binding activities relative to 3C and is devoid of RdRp activity. The origin of these differences is unclear, since crystal structure of 3CD revealed “beads-on-a-string” structure with no significant structural differences compared to the fully processed proteins. We performed molecular dynamics (MD) simulations on 3CD to investigate its conformational dynamics. A compact conformation of 3CD was observed that was substantially different from that shown crystallographically. This new conformation explained the unique properties of 3CD relative to the individual proteins. Interestingly, simulations of mutant 3CD showed altered interface. Additionally, accelerated MD simulations uncovered a conformational ensemble of 3CD. When we elucidated the 3CD conformations in solution using small-angle X-ray scattering (SAXS) experiments a range of conformations from extended to compact was revealed, validating the MD simulations. The existence of conformational ensemble of 3CD could be viewed as a way to expand the poliovirus proteome, an observation that may extend to other viruses. PMID:26610545

  10. Immune response to simultaneous administration of a combined measles, mumps and rubella vaccine with booster doses of diphtheria-tetanus and poliovirus vaccine.

    PubMed

    Giammanco, G; Li Volti, S; Salemi, I; Giammanco Bilancia, G; Mauro, L

    1993-03-01

    A combined vaccine against measles (Edmonston-Zagreb 19 strain), mumps (Rubini strain) and rubella (Wistar RA 27/3 strain) was administered to a group of 46 children aged 10-12 months simultaneously with booster doses of compulsory diphtheria-tetanus toxoid and oral poliovirus vaccine. A second group of 53 children aged 15-24 months who had received booster doses of the compulsory vaccines 5 to 12 months before was also vaccinated. The same seroconversion rates (100%) and similar antibody titers for rubella were observed in both groups. The same seroconversion rates for mumps (93%) and similar rates for measles (98 and 94%) were observed in the two groups. Significantly lower antibody titers for measles and mumps were found in the first group, but they were compensated by an earlier protection, a reduction of number of visits for immunization, costs for the community, and improvement in parental compliance. These results confirm that Edmonston-Zagreb 19 and Rubini strains are still immunogenic even when they are combined with Wistar RA 27/3 strain. Moreover, a long term follow-up in order to verify the persistence of protective antibody levels in both groups of children, could suggest that combined measles, mumps and rubella vaccine could be given earlier (at 10-12 months of age), simultaneously with booster doses of diphtheria and tetanus toxoid and of trivalent oral poliovirus vaccine. PMID:8519358

  11. Enhancement of the Replication of Hepatitis C Virus Replicons of Genotypes 1 to 4 by Manipulation of CpG and UpA Dinucleotide Frequencies and Use of Cell Lines Expressing SECL14L2 for Antiviral Resistance Testing.

    PubMed

    Witteveldt, Jeroen; Martin-Gans, Marion; Simmonds, Peter

    2016-05-01

    Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct-acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remain relatively unexplored, as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient-transfection format. In the current study, we have evaluated the effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCVs of different genotypes and evaluated the resulting assay formats for measurements of susceptibility to the DAA sofosbuvir. Removal of CpG and UpA dinucleotides from the luciferase gene used in HCV replicons of genotype 1b (Con1) and genotype 2a (JFH-1) achieved between 10- and 100-fold enhancement of replication over that of the wild type posttransfection. Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal. A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression. By combining these strategies, the 100- to 1,000-fold enhancement of replication allowed the susceptibility of all four genotypes to the RNA polymerase inhibitor sofosbuvir to be robustly determined in a transient-transfection assay format. These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs. PMID:26953209

  12. Depeptidization efforts on P[subscript 3]-P[prime subscript 2] [alpha]-ketoamide inhibitors of HCV NS3-4A serine protease: Effect on HCV replicon activity

    SciTech Connect

    Bogen, Stephane L.; Ruan, Sumei; Liu, Rong; Agrawal, Sony; Pichardo, John; Prongay, Andrew; Baroudy, Bahige; Saksena, Anil K.; Girijavallabhan, Viyyoor; Njoroge, F. George

    2008-06-30

    Depeptidization efforts of the P{sub 3}-P{sub 2} region of P{sub 3} capped {alpha}-ketoamide inhibitor of HCV NS3 serine protease 1 are reported. We clearly established that N-methylation of the P{sub 2} nitrogen and modification of the P{prime}{sub 2} carboxylic acid terminus were essential for activity in the replicon assay.

  13. Enhancement of the Replication of Hepatitis C Virus Replicons of Genotypes 1 to 4 by Manipulation of CpG and UpA Dinucleotide Frequencies and Use of Cell Lines Expressing SECL14L2 for Antiviral Resistance Testing

    PubMed Central

    Witteveldt, Jeroen; Martin-Gans, Marion

    2016-01-01

    Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct-acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remain relatively unexplored, as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient-transfection format. In the current study, we have evaluated the effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCVs of different genotypes and evaluated the resulting assay formats for measurements of susceptibility to the DAA sofosbuvir. Removal of CpG and UpA dinucleotides from the luciferase gene used in HCV replicons of genotype 1b (Con1) and genotype 2a (JFH-1) achieved between 10- and 100-fold enhancement of replication over that of the wild type posttransfection. Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal. A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression. By combining these strategies, the 100- to 1,000-fold enhancement of replication allowed the susceptibility of all four genotypes to the RNA polymerase inhibitor sofosbuvir to be robustly determined in a transient-transfection assay format. These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs. PMID:26953209

  14. Regional differences in the surface temperature of Naked Neck laying hens in a semi-arid environment

    NASA Astrophysics Data System (ADS)

    de Souza, João Batista Freire; de Arruda, Alex Martins Varela; Domingos, Hérica Girlane Tertulino; de Macedo Costa, Leonardo Lelis

    2013-05-01

    The aim of this study was to evaluate the regional differences in the surface temperature of Naked Neck hens that were subjected to different temperatures in a semi-arid environment. The surface temperature was measured in four body regions (face, neck, legs and feathered area) of 60 Naked Neck hens. The following environmental variables were measured at the center of the shed: the black globe temperature ( T G ), air temperature ( T A ), wind speed ( U) and relative humidity ( R H ). The T A was divided into three classes: 1 (24.0-26.0 °C), 2 (26.1-28.9 °C) and 3 (29.0-31.0 °C). An analysis of variance was performed by the least squares method and a comparison of the means by the Tukey-Kramer test. The results showed a significant effect of T A class, the body region and the interaction between these two effects on the surface temperature. There was no significant difference between the T A classes for the face and neck. The legs and feathered area showed significant differences between the T A classes. Regarding the effect of body regions within each T A class, there was a significant difference among all regions in the three T A classes. In all T A classes the neck had the highest average followed by the face and legs. The feathered area showed the lowest average of the different T A classes. In conclusion, this study showed that there are regional differences in the surface temperature of Naked Neck hens, with the legs acting as thermal windows.

  15. Regional differences in the surface temperature of Naked Neck laying hens in a semi-arid environment.

    PubMed

    de Souza, João Batista Freire; de Arruda, Alex Martins Varela; Domingos, Hérica Girlane Tertulino; de Macedo Costa, Leonardo Lelis

    2013-05-01

    The aim of this study was to evaluate the regional differences in the surface temperature of Naked Neck hens that were subjected to different temperatures in a semi-arid environment. The surface temperature was measured in four body regions (face, neck, legs and feathered area) of 60 Naked Neck hens. The following environmental variables were measured at the center of the shed: the black globe temperature (T G ), air temperature (T A ), wind speed (U) and relative humidity (R H ). The T A was divided into three classes: 1 (24.0-26.0 °C), 2 (26.1-28.9 °C) and 3 (29.0-31.0 °C). An analysis of variance was performed by the least squares method and a comparison of the means by the Tukey-Kramer test. The results showed a significant effect of T A class, the body region and the interaction between these two effects on the surface temperature. There was no significant difference between the T A classes for the face and neck. The legs and feathered area showed significant differences between the T A classes. Regarding the effect of body regions within each T A class, there was a significant difference among all regions in the three T A classes. In all T A classes the neck had the highest average followed by the face and legs. The feathered area showed the lowest average of the different T A classes. In conclusion, this study showed that there are regional differences in the surface temperature of Naked Neck hens, with the legs acting as thermal windows. PMID:22689146

  16. PacBio SMRT assembly of a complex multi-replicon genome reveals chlorocatechol degradative operon in a region of genome plasticity.

    PubMed

    Ricker, N; Shen, S Y; Goordial, J; Jin, S; Fulthorpe, R R

    2016-07-25

    We have sequenced a Burkholderia genome that contains multiple replicons and large repetitive elements that would make it inherently difficult to assemble by short read sequencing technologies. We illustrate how the integrated long read correction algorithms implemented through the PacBio Single Molecule Real-Time (SMRT) sequencing technology successfully provided a de novo assembly that is a reasonable estimate of both the gene content and genome organization without making any further modifications. This assembly is comparable to related organisms assembled by more labour intensive methods. Our assembled genome revealed regions of genome plasticity for further investigation, one of which harbours a chlorocatechol degradative operon highly homologous to those previously identified on globally ubiquitous plasmids. In an ideal world, this assembly would still require experimental validation to confirm gene order and copy number of repeated elements. However, we submit that particularly in instances where a polished genome is not the primary goal of the sequencing project, PacBio SMRT sequencing provides a financially viable option for generating a biologically relevant genome estimate that can be utilized by other researchers for comparative studies. PMID:27063562

  17. Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system.

    PubMed

    Moon, Ki-Beom; Lee, Jisu; Kang, Sebyung; Kim, Moonil; Mason, Hugh S; Jeon, Jae-Heung; Kim, Hyun-Soon

    2014-10-01

    Based on recent developments, virus-like particles (VLPs) are considered to be perfect candidates as nanoplatforms for applications in materials science and medicine. To succeed, mass production of VLPs and self-assembly into a correct form in plant systems are key factors. Here, we report expression of synthesized coat proteins of the three viruses, Brome mosaic virus, Cucumber mosaic virus, and Maize rayado fino virus, in Nicotiana benthamiana and production of self-assembled VLPs by transient expression system using agroinfiltration. Each coat protein was synthesized and cloned into a pBYR2fp single replicon vector. Target protein expression in cells containing p19 was fourfold higher than that of cells lacking p19. After agroinfiltration, protein expression was analyzed by SDS-PAGE and quantitative image analyzer. Quantitative analysis showed that BMVCP, CMVCP, and MRFVCP concentrations were 0.5, 1.0, and 0.8 mg · g(-1) leaf fresh weight, respectively. VLPs were purified by sucrose cushion ultracentrifugation and then analyzed by transmission electron microscopy. Our results suggested that BMVCP and CMVCP proteins expressed in N. benthamiana leaves were able to correctly self-assemble into particles. Moreover, we evaluated internal cavity accessibility of VLPs to load foreign molecules. Finally, plant growth conditions after agroinfiltration are critical for increasing heterologous protein expression levels in a transient expression system. PMID:24965559

  18. Sequence analysis and minimal replicon determination of a new haloarchaeal plasmid pHF2 isolated from Haloferax sp. strain Q22.

    PubMed

    Chen, Shaoxing; Wang, Chuangming; Xiang, Hua

    2016-01-01

    A new cryptic plasmid, pHF2 (2520 bp), was isolated from Haloferax sp. strain Q22 (CGMCC 1.15317), a haloarchaeal strain living in a subterranean halite deposit. Sequence analysis revealed that it is the smallest plasmid in the genus Haloferax so far, and three syntropic open reading frames (ORF1, ORF2, and ORF3) were identified on the same strand. ORF1 encodes a putative replication initiation protein (Rep). Three typical motifs (I, II, and III) were presented in the Rep proteins of rolling-circle replicating (RCR) plasmids. The amino acid sequence of the Rep protein is very similar to that of another haloarchaeal plasmid pNB101 in Natronobacterium sp. AS-7091 (coverage 97%, identity 56%). The minimal replicon (~1000 bp) of pHF2 was determined through the construction of a series of truncated plasmids. Interestingly, we also found that the incomplete rep gene still can drive plasmid replication. This plasmid has provided another valuable extra-chromosomal genetic resource, and deepened our knowledge in DNA replication. PMID:26601892

  19. Structure and Immunogenicity of Alternative Forms of the Simian Immunodeficiency Virus Gag Protein Expressed Using Venezuelan Equine Encephalitis Virus Replicon Particles

    PubMed Central

    Cecil, Chad; West, Ande; Collier, Martha; Jurgens, Christy; Madden, Victoria; Whitmore, Alan; Johnston, Robert; Moore, Dominic T.; Swanstrom, Ronald; Davis, Nancy L.

    2007-01-01

    Venezuelan equine encephalitis virus replicon particles (VRP) were engineered to express different forms of SIV Gag to compare expression in vitro, formation of intra- and extracellular structures and induction of humoral and cellular immunity in mice. The three forms examined were full-length myristylated SIV Gag (Gagmyr+), full-length Gag lacking the myristylation signal (Gagmyr-), or a truncated form of Gagmyr- comprising only the matrix and capsid domains (MA/CA). Comparison of VRP-infected primary mouse embryo fibroblasts, mouse L929 cells and primate Vero cells showed comparable expression levels for each protein, as well as extracellular virus-like particles (VRP-Gagmyr+), and distinctive cytoplasmic aggregates (VRP-Gagmyr-) with each cell type. VPR were used to immunize BALB/c mice, and immune responses were compared using an interferon (IFN)-γ ELISPOT assay and a serum antibody ELISA. Although all three VRP generated similar levels of IFN-γ-producing cells at 1 week post-boost, at 10 weeks post-boost the MA/CA-VRP-induced response was maintained at a significantly higher level relative to that induced by Gagmyr+-VRP. Antibody responses to MA/CA-VRP and Gagmyr+-VRP were not significantly different. PMID:17275057

  20. Naked-eye optical flash from gamma-ray burst 080319B: Tracing the decaying neutrons in the outflow

    SciTech Connect

    Fan Yizhong; Zhang Bing; Wei Daming

    2009-01-15

    For an unsteady baryonic gamma-ray burst (GRB) outflow, the fast and slow proton shells collide with each other and produce energetic soft gamma-ray emission. If the outflow has a significant neutron component, the ultrarelativistic neutrons initially expand freely until decaying at a larger radius. The late-time proton shells ejected from the GRB central engine, after powering the regular internal shocks, will sweep these {beta}-decay products and give rise to very bright UV/optical emission. The naked-eye optical flash from GRB 080319B, an energetic explosion in the distant Universe, can be well explained in this way.